WO1997011199A1 - Reactions d'acide nucleique - Google Patents

Reactions d'acide nucleique Download PDF

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Publication number
WO1997011199A1
WO1997011199A1 PCT/US1996/015199 US9615199W WO9711199A1 WO 1997011199 A1 WO1997011199 A1 WO 1997011199A1 US 9615199 W US9615199 W US 9615199W WO 9711199 A1 WO9711199 A1 WO 9711199A1
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single strand
duplex
nucleic acid
binding ligand
molecule
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PCT/US1996/015199
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English (en)
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WO1997011199A9 (fr
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Michael J. Lane
Albert S. Benight
Brian D. Faldasz
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Lane Michael J
Benight Albert S
Faldasz Brian D
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Priority to AU73685/96A priority Critical patent/AU7368596A/en
Publication of WO1997011199A1 publication Critical patent/WO1997011199A1/fr
Publication of WO1997011199A9 publication Critical patent/WO1997011199A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6823—Release of bound markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/682—Signal amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6862—Ligase chain reaction [LCR]

Definitions

  • This invention relates to the formation and dissolution of double stranded nucleic acid molecules and to the interactions between double and single stranded nucleic acid molecules and nucleic acid-binding ligands.
  • Ligands that bind to DNA span a broad range of sizes from small cations to large proteins and assembled protein aggregates.
  • a wide variety of experimental strategies have been employed to examine sequence specificity exhibited by ligands that interact with DNA. Sequence dependent variations in local conformation and charge configuration along DNA are thought to be the principle means by which ligands discriminate between various DNA sequences. Such discrimination can be divulged and quantitatively evaluated from sequence specific thermodynamic binding parameters evaluated in studies of ligand DNA complex formation. Double helical DNA structure is maintained by a number of forces.
  • DNA stability can be expressed as a number-weighted sum of the individual energies of two components, these being the "energies" of A-T and G-C bps For a specific sequence, i, this energy (the H-bond energy) can be designated
  • ⁇ G ⁇ -bond (') ⁇ S AT N AT T AT + ⁇ S G CNGC T GC ( 1 )
  • N A -f and NQC are tne numbers of A-T and G-C bps in the sequence and T A j and TQQ are the average melting temperatures of A-T (T-A) and G-C (C-G) bps Values of T j or TQQ evaluated from melting curve analysis of a variety of DNAs collected as a function of solvent environment provide the dependence of t m on solvent ionic strength The dependence of T j and TQQ on [Na+]22 was first reported by Frank-Kamenetski (Biopolymers 10:2623-24, 1971).
  • ⁇ S ⁇ and ⁇ S GG in eqn (1) are the average entropy changes associated with melting A-T or G-C bps.
  • Calorimetric and spectrophotometric melting studies of long DNA polymers of natural and synthetic origins have revealed the transition entropies of melting A-T and G-C bps are virtually independent of bp type (A-T or G-C), temperature, and only weakly dependent on solvent ionic strength over reasonable limits (15 m.M to 1.0 M NaCI) (Ref ?). Assuming only three preferred conformations are available for each nucleotide residue per bp, the transition entropy in forming a helix can be written as:
  • Restriction enzymes cleave duplex DNA at specific nucleotide sequences
  • the sequences flanking a restriction enzyme recognition site can influence the rate of restriction enzyme cleavage at the site (Aloyo et al , Biophys. J. 64 A280, 1993) Such effects occur while cleaving P4 phage DNA with the restriction enzyme EcoRI, suggesting that differences in DNA sequences flanking EcoRI sites account for observed differences in rates of cleavage (Goldstein et al , Virology 66 420-427, 1975) A large body of data regarding the sequence-dependent behavior of various restriction enzymes has appeared (Armstrong and Bauer, Nucl. Acids Res. 11 4109-4126, 1983, and Alves et al , Eur. J. Biochem.
  • the invention features methods of promoting a reaction between a duplex-binding ligand and a duplex nucleic acid molecule, the duplex nucleic acid molecule being formed from a first single strand nucleic acid molecule and a second single strand nucleic acid molecule, under conditions wherein the rate of duplex formation would be substantially less in the absence of the duplex-binding ligand
  • the method includes forming a reaction mixture including the first single strand nucleic acid molecule, the second single strand nucleic acid molecule, and the duplex-binding ligand under conditions wherein the rate of duplex formation would be substantially less in the absence of the duplex-binding ligand, the duplex-binding ligand being present at an amount, concentration, or chemical potential which results in the formation of a duplex at a rate which is substantially greater than the rate in the absence of the duplex-binding ligand, thereby allowing the reaction between the duplex and the duplex-binding ligand to proceed, and reacting the
  • reaction mixture comprising a plurality of the first single strand molecules, at least one of the second single strand molecules, a single strand binding ligand, and a duplex binding ligand comprising polymerase, an amount, concentration, or chemical potential of the single strand binding ligand and an amount, concentration, or chemical potential of the duplex-binding ligand being such that the following cycle of events can occur under isothermal conditions,
  • the invention features reaction mixtures comprising a single strand nucleic acid molecule, a second single strand nucleic acid molecule, a duplex and a duplex-binding ligand at an amount, concentration, or chemical potential which results in the formation of a duplex at a rate which is substantially greater than the rate in the absence ofthe duplex binding ligand.
  • the invention features reaction mixtures including: a plurality ofthe first single strand molecules, at least one second single strand molecule, a single strand binding ligand, and a duplex binding ligand comprising polymerase, an amount, concentration, or chemical potential of the single strand binding ligand and an amount, concentration, or chemical potential of the duplex-binding ligand being such that the following cycle of events can occur under isothermal conditions,
  • the invention features methods for detecting a single-stranded target nucleic acid, comprising (a) providing a reaction mixture which includes the target nucleic acid, a complementary single-stranded nucleic acid probe, the probe being present in molar excess relative to the target and having the structure [NA-- R-NA 2 ] n wherein NAi and NA 2 are DNA sequences, wherein R is a scissile nucleic acid linkage, and wherein n is an integer from 1 to 10, and a double strand binding ligand, the double strand binding ligand being present at a chemical potential sufficient to substantially increase the rate of duplex formation over what would be formed in the absence of the double strand binding ligand, and allowing target-probe duplex to form, (b) treating the target-probe duplex from step (a) so as to cleave the probe within a predetermined sequence ofthe scissile nucleic acid linkage
  • the methods and reagents of the invention can be used to determine the presence of a nucleic acid sequence present in a sample, and thus determine if a disease- related organism is present in a sample
  • the invention allows more sensitive and more selective detection of the nucleic acids of disease-related organisms, e.g.
  • Figure 1 is a diagram of a generalized reaction in which the chemical potentials ofa single strand binding agent and a duplex-binding agent provide for cycling between single strand and duplex states
  • Figure 2 is a diagram of a polymerase chain reaction driven by net chemical potential change, also referred to as isothermal PCR
  • Figure 3 is a depiction of the products of an isothermal PCR reaction as analyzed by gel electrophoresis
  • Figure 4 is a diagram of another generalized reaction in which the chemical potentials of a single strand binding agent and a duplex-binding agent (RNase H) provide for cycling between single strand and duplex states
  • RNase H duplex-binding agent
  • Tm refers to the midpoint of the duplex to single strand melting transition
  • the chemical potential of a reagent refers to the change in free energy of a reagent mix when the reagent is added to the mix
  • Chemical potential is a more exact measure of the activity of a species in a given reaction under a given set of conditions and takes into account considerations such as the number of sites a species can react with and whether all molecules of a species are available for reactions
  • Chemical potential of a species can usually be most directly manipulated by changing the concentrations of a species
  • the unit of chemical potential is free-energy, with units, e.g., of cal/mole or J/mole, but in the methods described herein determination of an absolute chemical potential is not required
  • Most of the methods disclosed herein require reaction mixtures in which the ratio of chemical potential between two species, or the difference between the chemical potential of two species, is required to be such that a given result, e.g., isothermal cycling, is achieved
  • a single strand binding ligand refers to a ligand which preferentially binds a single strand nucleic acid (as compared to the duplex or double stranded form of the nucleic acid)
  • Single strand binding ligands include, proteins, e.g., enzymes, as well as non-protein compounds
  • Single strand binding protein (SSB), the G-5 protein, the gene 32 protein, Rec A, and helicases are examples of single strand binding ligands
  • Single strand binding ligands stabilize the single stranded form and can thus be identified by their effect on the Tm of a duplex
  • a single strand binding ligand results in a decrease in the Tm of a duplex
  • duplex melting point determinations performed in the presence and absence of a test-compound will indicate if the test- compound is a single strand binding ligand for a sequence Before use in methods ofthe invention, the test-compound should be tested to determine if it has any unwanted effects on other
  • a double strand or duplex-binding ligand refers to a ligand which preferentially binds the double strand or duplex form of a nucleic acid in preference to single strands of the nucleic acid
  • Duplex-binding ligands can be proteins, e.g., enzymes, or non-protein compounds
  • suitable duplex- binding ligands include scissile-nucleic acid cleaving enzymes such as RNase H, DNA polymerase, actinomycin, and daunomycin are examples of double strand binding ligands
  • Double strand binding ligands stabilize the duplex form and can thus be identified by their effect on the Tm of a duplex
  • a double strand binding ligand results in an increase in the Tm of a duplex
  • duplex melting point determinations performed in the presence and absence of a test-compound will indicate if the test-compound is a double strand binding ligand for a sequence Before use, the test-compound should be
  • Purified nucleic acid refers to a purified DNA or RNA
  • Purified DNA refers to DNA that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (i.e., one at the 5' end and one at the 3' end) in the naturally-occurring genome of the organism from which the DNA is derived
  • the term includes, for example, a recombinant DNA which is inco ⁇ orated into a vector, e.g., into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other DNA sequences
  • Purified RNA refers to an RNA which is substantially free of another RNA sequence with which it is found in a cell which produces the RNA
  • Purified natural product is a product which is produced by an organism and which is substantially free of a macromolecule, e.g., a protein or a nucleic acid, with which it occurs in an organism from which it is derived
  • a product which does not naturally occur in living cells refers to a product which is not synthesized or produced by living cells or organisms
  • the invention features methods of promoting a reaction between a duplex-binding ligand and a duplex nucleic acid molecule, the duplex nucleic acid molecule being formed from a first single strand nucleic acid molecule and a second single strand nucleic acid molecule, under conditions wherein the rate of duplex formation would be substantially less in the absence of the duplex-binding ligand
  • Such methods include forming a reaction mixture including the first single strand nucleic acid molecule, the second single strand nucleic acid molecule, and the duplex-binding ligand under conditions wherein the rate of duplex formation would be substantially less in the absence of the duplex-binding ligand, the duplex-binding ligand being present at an amount, concentration, or chemical potential which results in the formation of a duplex at a rate which is substantially greater than the rate in the absence of the duplex-binding ligand, thereby allowing the reaction between the duplex and the duplex-binding ligand to proceed, and reacting the
  • the method is performed under isothermal conditions, the rate of duplex formation is increased by at least n-fold, wherein n is 2, 5, 10, 50, 100, 500, 10 3 , 10 4 , 10 5 , 10 6 , by the addition of duplex-binding ligand, the rate of duplex formation in the absence of duplex-binding ligand is substantially zero, at least one ofthe strand molecules is a purified nucleic acid molecule, the duplex-binding ligand is a purified natural product or a product which does not naturally occur in the living cells
  • the concentration, number of molecules of, or the chemical potential of, the duplex-binding ligand is greater than the concentration, number of molecules present, or chemical potential of the first single strand, the second single strand, both the first single strand and the second single strand, the duplex, or the combination of the first single strand, the second single strand, and the duplex, e.g., at least n-fold greater, wherein n is at least 1, 2, 5, 10, 25, 50, 100, 500, 10 3 , 10 4 , 10 5 or, 10 6
  • the first single strand molecule and the single strand molecule are present in substantially equal amounts or at substantially equal concentration, number, or chemical potential in the reaction mix, the first single strand molecule is a probe or primer molecule, the second single strand molecule is a target molecule, the first single strand molecule is a probe or primer molecule, the second single strand molecule is a target molecule and the ratio by weight, molarity, number, concentration, or chemical potential of the first sequence to the second sequence is less than or equal to 1 1, 2 1 , 5 1, 10 1, 25 1, 50 1, 100 1 , or 10 n 1 , wherein n is an integer between 3 and 10, inclusive, the free energy for the formation of the duplex from the first and the second sequence is decreased by at least n-fold, wherein n is 2, 5, 10, 50, 100, 500, 1,000, by addition of the duplex-binding ligand to the reaction mix, the ratio by weight, molarity, number, concentration, or chemical potential of the duplex-binding
  • the duplex-binding ligand comprises any of a compound which binds to a duplex nucleic acid in a sequence-specific way, a compound which binds to a duplex nucleic acid in a sequence-non-specific way, a protein, an enzyme, an enzyme which alters the structure of a duplex nucleic acid to which it binds, an enzyme which alters the structure of a duplex nucleic acid to which it binds by breaking or forming a covalent or non-covalent bond, between an atom of the nucleic acid and another atom, an enzyme which cleaves one or both strands of a duplex nucleic acid to which it binds, a restriction enzyme, a restriction endonuclease, an enzyme which methylates the duplex to which it binds, an enzyme which alkylates the duplex nucleic acid to which it binds, a nucleic acid ligase, e.g.
  • DNA ligase an enzyme which promotes or catalyzes the synthesis of a nucleic acid, a nucleic acid polymerase, a nucleic acid polymerase which requires a double stranded primer, a DNA polymerase, DNA polymerase I, Taq polymerase, an RNA polymerase, an enzyme which alters the primary or secondary structure of a duplex nucleic acid to which it binds, a topoisomerase, an enzyme which promotes or inhibits recombination, a DNA binding ligand, a mutagen, a compound which enhances the expression of a gene under the control of the duplex bound by a ligand, a compound which intercalates into a double stranded nucleic acid, a compound which, when contacted with a reaction mixture comprising a first single stranded nucleic acid and a second single stranded nucleic acid will accelerate the rate of duplex formation at least n-fold, wherein n is 2,
  • one or both single strand molecules are DNA, one or both single strand molecules are RNA, one single strand molecule is RNA and the other is DNA, one single strand molecule is a single stranded probe and the other single strand molecule is a target sequence which is to be cleared, detected, or amplified, one single strand molecule is a synthetic, purified natural, genetically engineered, or recombinant DNA or RNA molecule, and the other single strand molecule is a naturally occurring nucleic acid, e.g., a genomic molecule, or chromosome, e.g., a viral, bacterial, plant, or animal nucleic acid, one single strand molecule is a probe and one single strand molecule is the target sequence to be detected or amplified in an amplification reaction, or in a reaction in which amplification or detection is based on the amplification or detection ofthe probe or a fragment ofthe probe
  • the method further includes detecting a product of
  • the invention features methods of promoting a reaction between a duplex-binding ligand in a reaction mix and a duplex nucleic acid molecule, the duplex nucleic acid molecule formed from a first and a second single strand nucleic acid molecule, at a temperature substantially greater than the Tm of the duplex in the absence ofthe duplex-binding ligand
  • the method includes forming a reaction mixture comprising the first single strand nucleic acid molecule, the second single strand nucleic acid molecule, and the duplex-binding ligand at a temperature substantially greater the Tm of the duplex in the absence of the duplex-binding ligand, the duplex-binding ligand being present at an amount, concentration, or chemical potential which results in the formation of a duplex at the temperature, which thereby allows the reaction between the duplex and the duplex-binding ligand to proceed, and reacting duplex-binding ligand with the duplex at the temperature
  • the method is performed under isother
  • the concentration, number of molecules of, or the chemical potential of, the duplex-binding ligand is greater than the concentration, number of molecules present, or chemical potential of the first single strand, the second single strand, both the first single strand and the second single strand, the duplex, or the combination ofthe first single strand, the second single strand, and the duplex, e.g., at least n-fold greater, wherein n is at least 1 , 2, 5, 10, 25, 50, 100, 500, 10 3 , 10 4 , 10 5 , or 10 6
  • the first single strand molecule and the single strand molecule are present in substantially equal amounts or at substantially equal concentration, number, or chemical potentials in the reaction mix, the first single strand molecule is a probe or primer molecule, the second single strand molecule is a target molecule, the first single strand molecule is a probe or primer molecule, the second single strand molecule is a target molecule and the ratio by weight, molarity, number
  • the duplex-binding ligand comprises any of a compound which binds to a duplex nucleic acid in a sequence-specific way, a compound which binds to a duplex nucleic acid in a sequence-non-specific way, a protein, an enzyme, an enzyme which alters the structure of a duplex nucleic acid to which it binds, an enzyme which alters the structure of a duplex nucleic acid to which it binds by breaking or forming a covalent or non-covalent bond, between an atom of the nucleic acid and another atom, an enzyme which cleaves one or both strands of a duplex nucleic acid to which it binds, a restriction enzyme, an enzyme which cleaves a probe, such as the scissile linkage cleaving enzyme RNaseH, a restriction endonuclease, an enzyme which methylates the duplex to which it binds, an enzyme which alkylates the duplex nucleic acid to which
  • one or both single strand molecules are DNA, one or both single strand molecules are RNA, one single strand molecule is RNA and the other is DNA, one single strand molecule is a single stranded probe and the other single strand molecule is a target sequence which is to be cleared, detected, or amplified, one single strand molecule is a synthetic, purified natural, genetically engineered, or recombinant DNA or RNA molecule, and the other single strand molecule is a naturally occurring nucleic acid, e.g., a genomic molecule, or chromosome, e.g., a viral, bacterial, plant, or animal nucleic acid, one single strand molecule is a probe and one single strand molecule is the target sequence to be detected or amplified in an amplification reaction, or in a reaction in which amplification or detection is based on the amplification or detection ofthe probe or a fragment ofthe probe
  • the method further includes detecting a product ofthe reactions; the product is a nucleic acid sequence the formation of which is catalyzed in the reaction mix by the duplex-binding ligand, the duplex-binding ligand is a polymerase, e.g., a DNA polymerase, or a ligase
  • the invention features methods of performing a cycling reaction between a duplex-binding ligand and a duplex nucleic acid molecule formed from a first (e.g., a probe) and a second (e.g., a target) single strand nucleic acid molecule
  • a first e.g., a probe
  • a second e.g., a target
  • Such methods may comprise the general steps of
  • reaction mixture comprising a plurality of the first single strand molecules, at least one of the second single strand molecules, a single strand binding ligand, and a duplex strand binding ligand, the chemical potential of the single strand binding ligand and the chemical potential of the duplex strand binding ligand being such that the following cycle of events can occur under isothermal conditions,
  • n is at least 1, 2, 5, 10, 25, 50, 100, 500, 10 3 , 10 4 , 10*5, or lO ⁇ , of the above-described cycles to occur
  • duplex formation is modulated by inclusion of a single strand binding agent (SS- B) on the left side of the reaction that undergoes a binding reaction preferentially with the probe or the target contributing a free energy of duplex ligand complex ⁇ G P SS - B and ⁇ G T - SS - B AS discussed above, the free energy different between ⁇ G D E and ⁇ G SS - B , ⁇ Gr SS - B controls the direction of the cycling equilibrium by mediating duplex formation Note that if under the conditions in which the experiment is performed the free energies ⁇ G D E and ⁇ G SS - B , ⁇ G; SS-B for the opposing binding reactions are greater than the free energy of duplex formation, ⁇ Gn, only the enzyme concentrations, E and SS-B, determine the net direction ofthe reaction.
  • SS- B single strand binding agent
  • compositions of such enzymes constitute a nonobvious appropriate means for controlling the fidelity of duplex formation reactions
  • Fidelity of the reactions is independent of initial total DNA concentration when the duplex binding and single strand binding agents are present at high enough chemical potential which values are determined empirically or by other methods known in the art
  • a probe hybridizes to a target to form a duplex
  • the reaction has free energy ⁇ Go duplex
  • the probe includes a scissile link, which becomes susceptible to cleavage upon formation of a duplex
  • the problem can have an RNA-ase sensitive segment Formation of a duplex between such a probe and a single strand DNA molecule results in an RNA.DNA hybrid duplex.
  • RNA:DNA hybrids e.g., RJNAse H
  • Detection is based on this hybridization-specific cleavage RNAse binds the duplex with free energy ⁇ GDF to form a duplex RNAseH complex.
  • Cleavage results in a cleaved probe:target complex.
  • the cleavage probe disassociates from the target and the target can enter the cycle with a new, uncleaved probe.
  • Such methods may comprise the general steps of (a) hybridizing of the target nucleic acid sequence to a labeled nucleic acid probe to provide a probe:target nucleic acid sequence duplex; (b) cleaving only the labeled probe within the probe:target nucleic acid sequence duplex with an enzyme which causes selective probe cleavage resulting in duplex disassociation, leaving the target sequence intact; (c) recycling of the target nucleic acid sequence by repeating steps (a) and (b); and (d) detecting cleaved labeled probe, and thereby determining the presence ofthe target nucleic acid sequence.
  • single-stranded binding ligands may be utilized in the reaction mixture for methods of detecting a single-stranded target nucleic acid.
  • Such methods may comprise the general steps of (a) obtaining the single-stranded target nucleic acid, (b) forming a reaction mixture which includes the target nucleic acid and a complementary single-stranded nucleic acid probe under conditions which allow the target nucleic acid and the probe to hybridize to each other and form a double- stranded, target-probe complex, the probe being present in molar excess relative to the target and having the structure [NA r R-NA 2 ] n wherein NA- and NA 2 are DNA sequences, wherein R is a scissile nucleic acid linkage, and wherein n is an integer from 1 to 10, (c) treating the double-stranded, target-probe complex from step (b) so as to cleave the probe within a predetermined sequence of the scissile nucleic acid linkage and thereby form at
  • the single strand binding ligand is a purified natural product or a non-naturally occurring product; the concentration, number of molecules present, or chemical potential of the single strand binding ligand is greater than the concentration, number of molecules present, or chemical potential of the first single strand, the second single strand, both the first single strand and the second single strand, the duplex, or the combination of the first single strand, the second single strand, and the duplex, e.g., at least n-fold greater, wherein n is at least 1, 2, 5, 10, 25, 50, 100, 500, 10 3 , 10 4 , 10 5 or 10 6
  • the single strand binding ligand comprises any of. a compound which binds to a single strand nucleic acid in a sequence- specific way; a compound which binds to a single strand nucleic acid in a sequence-non ⁇ specific way; a protein; an enzyme; an enzyme which alters the structure ofa nucleic acid to which it binds; an enzyme which alters the structure of a nucleic acid to which it binds by breaking or forming a covalent or non-covalent bond, between an atom ofthe nucleic acid and another atom; an enzyme which cleaves a nucleic acid to which it binds; a restriction enzyme; a restriction endonuclease; an enzyme which methylates the nucleic acid to which it binds; an enzyme which alkylates the duplex nucleic acid to which it binds; an enzyme which alters the primary or secondary structure of a duplex nucleic acid to which it binds, an enzyme which promotes or inhibit
  • the concentration, number of molecules present, or chemical potential of the single strand binding ligand is no more than 10 times the concentration, number of molecules present, or chemical potential of the duplex-binding ligand, the concentration, number of molecules present, or chemical potential of the single strand binding ligand is no less than one-tenth the concentration, number of molecules present, or chemical potential of the duplex-binding ligand, the concentration, number of molecules present, or chemical potential of the duplex-binding ligand is no more than 10 times the concentration, number of molecules present, or chemical potential of the single strand binding ligand, the concentration, number of molecules present, or chemical potential of the duplex-binding ligand is no less than one- tenth the concentration, number of molecules present, or chemical potential of the single strand binding ligand In other preferred embodiments the concentration, number of molecules present, or chemical potential of the duplex-binding ligand and the concentration, number of molecules present, or chemical potential ofthe single strand binding ligand are such that the rate of formation
  • the method is performed under isothermal conditions, the temperature is above the Tm, the temperature is at least n C°, wherein n is 5, 10, 20, 30, 40, 50, 60, 70, or 80, above the Tm, at least one ofthe strand molecules is a purified nucleic acid molecule
  • the number of molecules of, or the chemical potential of, the duplex-binding ligand is greater than the concentration, number of molecules present, or chemical potential of the first single strand, the second single strand, both the first single strand and the second single strand, the duplex, or the combination of the first single strand, the second single strand, and the duplex, e.g., at least n-fold greater, wherein n is at least 1 , 2, 5, 10, 25, 50, 100, 500, 10 3 , 10 4 , 10 5 or 10 6
  • the first single strand molecule and the second single strand molecule are present in substantially equal amounts or at substantially equal amount, concentration, or chemical potential in the reaction mix, the first single strand molecule is a probe or primer molecule, the second single strand molecule is a target molecule, the first single strand molecule is a probe or primer molecule, the second single strand molecule is a target molecule and the ratio by weight, molarity, number, concentration, or chemical potentials of the first sequence to the second sequence is less than or equal to 1 1 , 2 1 , 5 1 , 10 1 , 25 1 , 50 1 , 100 1 , or 10 n 1, wherein n is an integer between 3 and 10, inclusive, the ratio by weight, molarity, number, concentration, or chemical potentials of the duplex-binding ligand to the single strand in the highest concentration is greater than 1 1 , 2 1 , 5 1 , 10 1, 25 1, 50 1, 100 1, or 10 n 1, wherein n is an integer between 3 and
  • one or both single strand molecules are DNA, one or both single strand molecules are RNA, one single strand molecule is RNA and the other is DNA, one single strand molecule is a single stranded probe and the other single strand molecule is a target sequence which is to be cleared, detected, or amplified, one single strand molecule is a synthetic, purified natural, genetically engineered, or recombinant DNA or RNA molecule, and the other single strand molecule is a naturally occurring nucleic acid, e.g., a genomic molecule, or chromosome, e.g., a viral, bacterial, plant, or animal nucleic acid, one single strand molecule is a probe and one single strand molecule is the target sequence to be detected or amplified in an amplification reaction, or in a reaction in which amplification or detection is based on the amplification or detection of the probe or a fragment of the probe
  • such methods further include detecting a product ofthe reactions, the product is a nucleic acid sequence the formation of which is catalyzed in the reaction mix by the duplex-binding ligand, the duplex-binding ligand is a polymerase, e.g., a DNA polymerase, or a ligase, the single strand binding ligand is present in sufficient concentration, number of molecules, or chemical potential such that there is substantially no non-specific hybridization ofthe first single strand to a sequence other than the second single strand
  • the invention features methods of performing a polymerase reaction using as a double stranded substrate a duplex nucleic acid molecule formed from a first (e.g., a probe) and a second (e.g., a target) single strand nucleic acid molecule
  • the method includes (1) forming a reaction mixture comprising a plurality of the first single strand molecules, at least one of the second single strand molecules, a single strand binding ligand, and a duplex-binding ligand comprising polymerase, an amount, concentration, or chemical potential of the single strand binding ligand and an amount, concentration, or chemical potential of the duplex-binding ligand being such that the following cycle of events can occur under isothermal conditions,
  • n is at least 1 , 2, 5, 10, 25, 50, 100,
  • the single strand binding ligand is a purified natural product or a non-naturally occurring product DEFINE
  • the concentration, number of molecules present, or chemical potential of the single strand binding ligand is greater than the concentration, number of molecules present, or chemical potential of the first single strand, the second single strand, both the first single strand and the second single strand, the duplex, or the combination of the first single strand, the second single strand, and the duplex, e.g., at least n-fold greater, wherein n is at least 1 , 2, 5, 10, 25, 50, 100, 500, 10 3 , 10 4 , 10 5 or 10 6
  • the single strand binding ligand comprises any of a compound which binds to a single strand nucleic acid in a sequence- specific way, a compound which binds to a single strand nucleic acid in a sequence-non ⁇ specific way, a protein, an enzyme, an enzyme which alters the structure ofa nucleic acid to which it binds, an enzyme which alters the structure of a nucleic acid to which it binds by breaking or forming a covalent or non-covalent bond between an atom of the nucleic acid and another atom, an enzyme which cleaves a nucleic acid to which it binds, a restriction enzyme, a restriction endonuclease, an enzyme which methylates the nucleic acid to which it binds, an enzyme which alkylates the duplex nucleic acid to which it binds, an enzyme which alters the primary or secondary structure of a duplex nucleic acid to which it binds; an enzyme which promotes or inhibits
  • n 2, 5, 10, 50, 100, 500, 10 3 , 10 4 , 10 5 , 10 6 , a compound which, when contacted with a reaction mixture, will decrease the free energy of single strand formation by at least n-fold, wherein n is 2, 5, 10, 50, 100, 500, 10 3 , 10 4 , 10 5 , 10 6
  • the concentration, number of molecules present, or chemical potential of the single strand binding ligand is no more than 10 times the concentration, number of molecules present, or chemical potential of the duplex-binding ligand, the concentration, number of molecules present, or chemical potential of the single strand binding ligand is no less than one-tenth the concentration, number of molecules present, or chemical potential of the duplex-binding ligand, the concentration, number of molecules present or chemical potential of the duplex-binding ligand is no more than 10 times the concentration, number of molecules present, or chemical potential of the single strand binding ligand, the concentration, number of molecules present, or chemical potential ofthe duplex-binding ligand is no less than one- tenth the concentration, number of molecules present, or chemical potential of the single strand binding ligand In other preferred embodiments the concentration, number of molecules present, or chemical potential of the duplex-binding ligand and the concentration, number of molecules present, or chemical potential ofthe single strand binding ligand are such that the rate of formation of
  • the method is performed under isothermal conditions, the temperature is above the Tm, the temperature is at least n C°, wherein n is 5, 10, 20, 30, 40, 50, 60, 70, or 80, above the Tm, at least one ofthe single strand molecules is a purified nucleic acid molecule
  • the number of molecules of, or the chemical potential of, the duplex-binding ligand is greater than the concentration, number of molecules present, or chemical potential of the first single strand, the second single strand, both the first single strand and the second single strand, the duplex, or the combination of the first single strand, the second single strand, and the duplex, e.g., at least n-fold greater, wherein n is at least 1, 2, 5, 10, 25, 50, 100, 500, 10 3 , 10 4 , 10 5 or 10 6 .
  • the first single strand molecule and the single strand molecule are present in substantially equal amounts or at substantially equal an amount, concentration, or chemical potential in the reaction mix, the first single strand molecule is a probe or primer molecule, the second single strand molecule is a target molecule
  • the first single strand molecule is a probe or primer molecule
  • the second single strand molecule is a target molecule and the ratio by weight, molarity, number, concentration, or chemical potentials of the first sequence to the second sequence is less than or equal to 1 1 , 2 1 , 5 1 , 10 1, 25 1, 50 1, 100 * 1, or 10 n 1, wherein n is an integer between 3 and 10, inclusive
  • the ratio by weight, molarity, number, concentration, or chemical potentials of the duplex-binding ligand to the single strand in the highest concentration is greater than 1 1, 2 1, 5 1 , 10 1 , 25 1 , 50 1, 100 1, or 10 n 1, wherein n is an integer between 3 and 10, inclusive
  • the duplex-binding ligand in addition to a polymerase, comprises any of a compound which binds to a duplex nucleic acid in a sequence-specific way, a compound which binds to a duplex nucleic acid in a sequence- non-specific way, a protein, an enzyme, an enzyme which alters the structure of a duplex nucleic acid to which is binds, an enzyme which alters the structure of a duplex nucleic acid to which it binds by breaking or forming a covalent or non-covalent bond, between an atom of the nucleic acid and another atom, an enzyme which cleaves one or both strands of a duplex nucleic acid to which it binds, a restriction enzyme, a restriction endonuclease, an enzyme which in methylates the duplex to which it binds, an enzyme which alkylates the duplex nucleic acid to which it binds, a nucleic acid ligase, e
  • one or both single strand molecules are DNA, one or both single strand molecules are R A, one single strand molecule is RNA and the other is DNA, one single strand molecule is a single stranded probe and the other single strand molecule is a target sequence which is to be cleared, detected, or amplified, one single strand molecule is a synthetic, purified natural, genetically engineered, or recombinant DNA or RNA molecule, and the other single strand molecule is a naturally occurring nucleic acid, e g , a genomic molecule, or chromosome, e.g , a viral, bacterial, plant, or animal nucleic acid, one single strand molecule is a probe and one single strand molecule is the target sequence to be detected or amplified in an amplification reaction, or in a reaction in which amplification or detection is based on the amplification or detection ofthe probe or a fragment ofthe probe
  • the method further including detecting a product ofthe reactions, the product is a nucleic acid sequence the formation of which is catalyzed in the reaction mix by the duplex-binding ligand, the duplex-binding ligand is a DNA polymerase, the single strand binding ligand is present in sufficient concentration, number of molecules, or chemical potential such that there is substantially no non ⁇ specific hybridization of the first single strand to a sequence other than the second single strand
  • the invention features methods of cyclically reacting a duplex-binding ligand with a duplex formed by a hybridization of a first single strand molecule (e g , a probe) molecule to a second single strand molecule (e.g , a target)
  • a first single strand molecule e.g , a probe
  • a second single strand molecule e.g , a target
  • a reaction mixture comprising the first single strand molecule, the second single strand molecule, a single strand binding ligand, and a duplex-binding ligand, the chemical potential of the duplex-binding ligand being sufficient to cause the formation of duplex, and the chemical potential of the single strand binding ligand being such that duplex dissociation reaction occurs concurrently with the formation reaction, the chemical potentials being such that the rate of duplex formation and the rate of duplex dissociation are such that a cycle of (duplex-binding ligand duplex-bindmg)- (duplex d ⁇ ssoc ⁇ at ⁇ on)-(duplex-b ⁇ nd ⁇ ng ligand duplex-binding) could occur under isothermal conditions, and allowing the cycle to proceed As discussed above, a wide va ⁇ ety of cycling or catalytic reactions may be accomplished in this manner
  • the cycle can occur least n times under isothermal conditions, wherein n is at least 1 , 2, 5, 10, 25, 50, 100, 500, 10 3 , 10 4 , 10 5 , or 10 6
  • the single strand binding ligand is a purified natural product or a non-naturally occurring product
  • the concentration, number of molecules present, or chemical potential of the single strand binding ligand is greater than the concentration, number of molecules present, or chemical potential of the first single strand, the second single strand, both the first single strand and the second single strand, the duplex, or the combination of the first single strand, the second single strand, and the duplex, e.g., at least n-fold greater, wherein n is at least 1 , 2, 5, 10, 25, 50, 100, 500, 10 3 , 10 4 , 10 5 , or 10 6
  • the single strand binding ligand comprises any of a compound which binds to a single strand nucleic acid in a sequence- specific way, a compound which binds to a single strand nucleic acid in a sequence-non ⁇ specific way, a protein, an enzyme, an enzyme which alters the structure ofa nucleic acid to which it binds, an enzyme which alters the structure of a nucleic acid to which it binds by breaking or forming a covalent or non-covalent bond, between an atom of the nucleic acid and another atom, an enzyme which cleaves a nucleic acid to which it binds, a restriction enzyme, a restriction endonuclease, an enzyme which methylates the nucleic acid to which it binds, an enzyme which alkylates the duplex nucleic acid to which it binds, an enzyme which alters the primary or secondary structure of a duplex nucleic acid to which it binds, an enzyme which promotes or
  • the concentration, number of molecules present, or chemical potential of the single strand binding ligand is no more than 10 times the concentration, number of molecules present, or chemical potential of the duplex-binding ligand, the concentration, number of molecules present, or chemical potential of the single strand binding ligand is no less than one-tenth the concentration, number of molecules present, or chemical potential of the duplex-binding ligand, the concentration, number of molecules present, or chemical potential of the duplex-binding ligand is no more than 10 times the concentration, number of molecules present, or chemical potential of the single strand binding ligand, the concentration, number of molecules present, or chemical potential of the duplex-binding ligand is no less than one- tenth the concentration, number of molecules present, or chemical potential of the single strand binding ligand
  • the concentration, number of molecules present, or chemical potential of the duplex-binding ligand and the concentration, number of molecules present, or chemical potential ofthe single strand binding ligand are such that the rate of formation of duplex is substantially equal to the rate of formation of single strands
  • the concentration, number of molecules present, or chemical potential of the duplex-binding ligand and the concentration, number of molecules present, or chemical potential ofthe single strand binding ligand are such that the rate of formation of duplex is no more than 2, 5, 10, 20, 50, 100, 10 J or 10 4 times the rate of formation of single strands
  • the concentration, number of molecules present, or chemical potential of the duplex-binding ligand and the concentration, number of molecules present, or chemical potential of the single strand binding ligand are such that the rate of formation of single strands is no more than 2, 5, 10, 20, 50, 100, 10 3 or 10 4 times the rate of formation of duplex
  • the method is performed under isothermal conditions, the temperature is above the Tm, the temperature
  • the number of molecules of, or the chemical potential of, the duplex-binding ligand is greater than the concentration, number of molecules present, or chemical potential of the first single strand, the second single strand, both the first single strand and the second single strand, the duplex, or the combination of the first single strand, the second single strand, and the duplex, e.g., at least n-fold greater, wherein n is at least 1, 2, 5, 10, 25, 50, 100, 500, 10 3 , 10 4 , 10 5 , or 10 6
  • the first single strand molecule and the single strand molecule are present in substantially equal amounts or at substantially equal chemical potentials in the reaction mix
  • the first single strand molecule is a probe or primer molecule
  • the second single strand molecule is a target molecule
  • the first single stand molecule is a probe or primer molecule
  • the second single strand molecule is a target molecule
  • the ratio by weight, molarity, number, concentration, or chemical potentials of the first sequence to the second sequence is less than or equal to 1.1, 2 1, 5 1 , 10 1, 25 1 , 50 1, 100 1, or 10 n 1 , wherein n is an integer between 3 and 10, inclusive
  • the ratio by weight, molarity, number, concentration, or chemical potentials of the duplex-binding ligand to the single strand in the highest concentration is greater than 1 1, 2 1, 5 1, 10 1, 25 1 , 50 1, 100 1 , or 10 n 1 , wherein n is an integer between 3 and 10, inclusive
  • the duplex-binding ligand comprises any of a compound which binds to a duplex nucleic acid in a sequence-specific way, a compound which binds to a duplex nucleic acid in a sequence-non-specific way, a protein, an enzyme, an enzyme which alters the structure of a duplex nucleic acid to which is binds, an enzyme which alters the structure of a duplex nucleic acid to which it binds by breaking or forming a covalent or non-covalent bond, between an atom of the nucleic acid and another atom; an enzyme which cleaves one or both strands of a duplex nucleic acid to which it binds, a restriction enzyme; a restriction endonuclease; an enzyme which methylates the duplex to which it binds; an enzyme which alkylates the duplex nucleic acid to which it binds; a nucleic acid ligase, e.g., DNA ligase;
  • DNA, one or both single strand molecules are RNA; one single strand molecule is RNA and the other is DNA; one single strand molecule is a single stranded probe and the other single strand molecule is a target sequence which is to be cleared, detected, or amplified, one single strand molecule is a synthetic, purified natural, genetically engineered, or recombinant DNA or RNA molecule, and the other single strand molecule is a naturally occurring nucleic acid, e.g., a genomic molecule, or chromosome, e.g., a viral, bacterial, plant, or animal nucleic acid; one single strand molecule is a probe and one single strand molecule is the target sequence to be detected or amplified in an amplification reaction, or in a reaction in which amplification or detection is based on the amplification or detection ofthe probe or a fragment ofthe probe.
  • a naturally occurring nucleic acid e.g., a genomic molecule, or chromos
  • the method further including detecting a product of the cyclic reactions;
  • the product is a nucleic acid sequence the formation of which is catalyzed in the reaction mix by the duplex-binding ligand
  • the duplex-binding ligand is a polymerase, e.g., a DNA polymerase, or a ligase
  • the single strand binding ligand is present in sufficient concentration, number of molecules, or chemical potential such that there is substantially no non-specific hybridization of the first single strand to a sequence other than the second single strand
  • the invention features a method of detecting the hybridization of a first single strand molecule (e.g., a probe) to a second single strand molecule (e.g., a target) comprising forming a reaction mixture comprising the first molecule, the second molecule, a single strand binding ligand, and a duplex-binding ligand comprising a polymerase, a ligase, or other sequence forming enzyme, an amount, concentration, or chemical potential ofthe duplex-binding ligand being sufficient to cause the formation of duplex, and an amount, concentration, or chemical potential of the single strand binding ligand being such that a predetermined level of duplex dissociation reaction occurs, the rate of duplex formation and the rate of duplex dissociation being such that a cycle of duplex-binding ligand catalyzed formation-duplex dissociation-duplex-binding ligand catalyzed formation which can occur under isothermal conditions occurs, allowing the reaction to proceed in the
  • the single strand molecule is labeled with a detectable marker when it hybridizes to the second strand, the single strand molecule is labeled after hybridizing to the second molecule, the NTPs are labeled and a labeled extension product is detected, the single strand binding ligand is a purified natural product or a non-naturally occurring product DEFINE, the concentration, number of molecules present, or chemical potential of the single strand binding ligand is greater than the concentration, number of molecules present, or chemical potential ofthe first single strand, the second single strand, both the first single strand and the second single strand, the duplex, or the combination of the first single strand, the second single strand, and the duplex, e.g., at least n-fold greater, wherein n is at least 1, 2, 5, 10, 25, 50, 100, 500, 10 3 , 10 4 , 10 s , or 10 6 .
  • the single strand binding ligand comprises any of a compound which binds to a single strand nucleic acid in a sequence- specific way, a compound which binds to a single strand nucleic acid in a sequence-non ⁇ specific way, a protein, an enzyme, an enzyme which alters the structure of a nucleic acid to which it binds, an enzyme which alters the structure of a nucleic acid to which it binds by breaking or forming a covalent or non-covalent bond, between an atom ofthe nucleic acid and another atom, an enzyme which cleaves a nucleic acid to which it binds, a restriction enzyme, a restriction endonuclease, an enzyme which methylates the nucleic acid to which it binds, an enzyme which alkylates the duplex nucleic acid to which it binds; an enzyme which alters the primary or secondary structure of a duplex nucleic acid to which it binds; an enzyme which promotes or inhibit
  • the concentration, number of molecules present, or chemical potential of the single strand binding ligand is no more than 10 times the concentration, number of molecules present, or chemical potential of the duplex-binding ligand; the concentration, number of molecules present, or chemical potential of the single strand binding ligand is no less than one-tenth the concentration, number of molecules present, or chemical potential of the duplex-binding ligand; the concentration, number of molecules present, or chemical potential of the duplex-binding ligand is no more than 10 times the concentration, number of molecules present, or chemical potential of the single strand binding ligand, the concentration, number of molecules present, or chemical potential ofthe duplex-binding ligand is no less than one- tenth the concentration, number of molecules present, or chemical potential of the single strand binding ligand
  • the concentration, number of molecules present, or chemical potential of the duplex-binding ligand and the concentration, number of molecules present, or chemical potential ofthe single strand binding ligand are such that the rate of formation of duplex is substantially equal to the rate of formation of single strands; the concentration, number of molecules present, or chemical potential of the duplex-binding ligand and the concentration, number of molecules present, or chemical potential of the single strand binding ligand are such that the rate of formation of duplex is no more than 2, 5, 10, 20, 50, 100, 10 3 or 10 4 times the rate of formation of single strands, the concentration, number of molecules present, or chemical potential of the duplex-binding ligand and the concentration, number of molecules present, or chemical potential of the single strand binding ligand are such that the rate of formation of single strands is no more than 2, 5, 10, 20, 50, 100, 10 3 or 10 4 times the rate of formation of duplex
  • the method is performed under isothermal conditions; the temperature is above the Tm, the temperature
  • the first single strand molecule and the second single strand molecule are present in substantially equal amounts or at substantially equal an amount, concentration, or chemical potentials in the reaction mix, the first single strand molecule is a probe or primer molecule, the second single strand molecule is a target molecule, the first single strand molecule is a probe or primer molecule, the second single strand molecule is a target molecule and the ratio by weight, molarity, number, concentration, or chemical potentials of the first sequence to the second sequence is less than or equal to 1 1 , 2 1, 5 1 , 10 1, 25 1, 50 1, 100 1, or 10 n l, wherein n is an integer between 3 and 10, inclusive, the ratio by weight, molarity, number, concentration, or chemical potentials of the duplex-binding ligand to the single strand in the highest concentration is greater than 1 1 , 2 1 , 5 1 , 10 I , 25 1 , 50 1, 100 1, or 10 n . l, wherein n is an integer between
  • the duplex-binding ligand comprises any of a compound which binds to a duplex nucleic acid in a sequence-specific way, a compound which binds to a duplex nucleic acid in a sequence-non-specific way; a protein, an enzyme, an enzyme which alters the structure of a duplex nucleic acid to which is binds, an enzyme which alters the structure of a duplex nucleic acid to which it binds by breaking or forming a covalent or non-covalent bond, between an atom of the nucleic acid and another atom, an enzyme which cleaves one or both strands of a duplex nucleic acid to which it binds, a restriction enzyme, a restriction endonuclease, an enzyme which methylates the duplex to which it binds, an enzyme which alkylates the duplex nucleic acid to which it binds, a nucleic acid ligase, e.g., DNA ligase,
  • one or both single strand molecules are DNA, one or both single strand molecules are RNA, one single strand molecule is RNA and the other is DNA, one single strand molecule is a single stranded probe and the other single strand molecule is a target sequence which is to be cleared, detected, or amplified, one single strand molecule is a synthetic, purified natural, genetically engineered, or recombinant DNA or RNA molecule, and the other single strand molecule is a naturally occurring nucleic acid, e.g., a genomic molecule, or chromosome, e.g., a viral, bacterial, plant, or animal nucleic acid, one single strand molecule is a probe and one single strand molecule is the target sequence to be detected or amplified in an amplification reaction, or in a reaction in which amplification or detection is based on the amplification or detection ofthe probe or a fragment of the probe
  • the method further including detecting a product ofthe reactions, the product is a nucleic acid sequence the formation of which is catalyzed in the reaction mix by the duplex-binding ligand, the single strand binding ligand is present in sufficient concentration, number of molecules, or chemical potential such that there is substantially no non-specific hybridization of the first single strand to a sequence other than the second single strand
  • the invention features methods of promoting a reaction between a single strand binding ligand and a single strand nucleic acid molecule, the single strand nucleic acid molecule being formed from a duplex comprising the first single strand nucleic acid molecule and a second single strand nucleic acid molecule, under conditions wherein the rate of single strand formation would be substantially less in the absence ofthe duplex-binding ligand
  • the method includes forming a reaction mixture comprising the first single strand nucleic acid molecule, the second single strand nucleic acid molecule, and the single strand binding ligand under conditions wherein the rate of single strand formation would be substantially less in the absence of the single strand binding ligand, the single strand binding ligand being present at a chemical potential which results in the formation of a single strand at a rate which is substantially greater than the rate in the absence of the single strand binding ligand, thereby allowing the reaction between the single strand and the single strand binding ligand to proceed, and
  • the method is performed under isothermal conditions, the rate of single strand formation is increased by at least n-fold, wherein n is 2, 5, 10, 50, 100, 500, 10 3 , 10 4 , 10 5 , 10 6 , by the addition of single strand binding ligand, the rate of single strand formation in the absence of single strand binding ligand is substantially zero, at least one of the single strand molecules is a purified nucleic acid molecule, the single strand binding ligand is a purified natural product or a product which does not naturally occur in the living cells
  • the concentration, number of molecules of, or the chemical potential of, the single strand binding ligand is greater than the concentration, number of molecules present, or chemical potential of the first single strand, the second single strand, both the first single strand and the second single strand, the duplex, or the combination of the first single strand, the second single strand, and the duplex, e.g., at least n-fold greater, wherein n is 2, 5, 10, 50, 100
  • the first single strand molecule and the second single strand molecule are present in substantially equal amounts or at substantially equal concentration, number, or chemical potentials in the reaction mix
  • the first single strand molecule is a probe or primer molecule
  • the second single strand molecule is a target molecule
  • the first single strand molecule is a probe or primer molecule
  • the second single strand molecule is a target molecule
  • the ratio by weight, molarity, number, concentration, or chemical potential of the first sequence to the second sequence is less than or equal to 1 1 , 2 1 , 5 1 , 10 1 , 25 1, 50 1, 100 1, or 10 n 1, wherein n is an integer between 3 and 10, inclusive, the free energy for the formation of the single strand from the duplex is decreased by at least n-fold, wherein n is 2, 5, 10, 50, 100, 500, 1,000, by addition of the single strand binding ligand to the reaction mix, the ratio by weight, molarity, number, concentration, or chemical potential of the single strand binding
  • one or both single strand molecules are DNA; one or both single strand molecules are RNA, one single strand molecule is RNA and the other is DNA; one single strand molecule is a single stranded probe and the other single strand molecule is a target sequence which is to be cleared, detected, or amplified; one single strand molecule is a synthetic, purified natural, genetically engineered, or recombinant DNA or RNA molecule, and the other single strand molecule is a naturally occurring nucleic acid, e.g., a genomic molecule, or chromosome, e.g., a viral, bacterial, plant, or animal nucleic acid, one single strand molecule is a probe and one single strand molecule is the target sequence to be detected or amplified in an amplification reaction, or in a reaction in which amplification or detection is based on the amplification or detection ofthe probe or a fragment ofthe probe
  • the method further including detecting a product ofthe reactions; the product is a nucleic acid sequence the formation of which is catalyzed in the reaction mix by the single strand binding ligand
  • the invention features methods of promoting a reaction between a single strand binding ligand in a reaction mix and a single strand nucleic acid molecule, the single strand nucleic acid molecule formed from a duplex comprising a first and a second single strand nucleic acid molecule, at a temperature substantially less than the Tm of the duplex in the absence of the single strand binding ligand
  • the method includes: forming a reaction mixture comprising the first single strand nucleic acid molecule, the second single strand nucleic acid molecule, and the single strand binding ligand at a temperature substantially less the Tm of the duplex in the absence of the duplex-binding ligand, the single strand binding ligand being present at an amount, concentration, or chemical potential which results in the formation of a single strand at the temperature, which thereby allows the reaction between the single strand and the single stand binding ligand to proceed, and reacting the single strand binding ligand with the single strand at the temperature
  • Such methods may be
  • the concentration, number of molecules of, or the chemical potential of, the single strand binding ligand is greater than the concentration, number of molecules present, or chemical potential of the first single strand, the second single strand, both the first single strand and the second single strand, the duplex, or the combination ofthe first single strand, the second single strand, and the duplex, e.g., at least n-fold greater, wherein n is at least 1, 2, 5, 10, 25, 50, 100, 500, 10 3 , 10 4 , 10 5 , or 10 6
  • the first single strand molecule and the second single strand molecule are present in substantially equal amounts or at substantially equal concentration, number, or chemical potentials in the reaction mix, the first single strand molecule is a probe or primer molecule, the second single strand molecule is a target molecule, the first single strand molecule is a probe or primer molecule, the second single strand molecule is a target molecule and the ratio by weight, molarity, number, concentration
  • the single strand binding ligand comprises any of a compound which binds to a single strand nucleic acid in a sequence- specific way; a compound which binds to a single strand nucleic acid in a sequence-non ⁇ specific way; a protein; an enzyme, an enzyme which alters the structure of a single strand nucleic acid to which is binds, an enzyme which alters the structure of a single strand nucleic acid to which it binds by breaking or forming a covalent or non-covalent bond, between an atom of the nucleic acid and another atom, an enzyme which cleaves the nucleic acid to which it binds, a restriction enzyme, a restriction endonuclease, an enzyme which alkylates the duplex nucleic acid to which it binds, a nucleic acid ligase, e.g., DNA ligase, an enzyme which promotes or catalyzes the synthesis of a nucleic acid, an enzyme which alters
  • one or both single strand molecules are DNA, one or both single strand molecules are RNA, one single strand molecule is RNA and the other is DNA, one single strand molecule is a single stranded probe and the other single strand molecule is a target sequence which is to be cleared, detected, or amplified; one single strand molecule is a synthetic, purified natural, genetically engineered, or recombinant DNA or RNA molecule, and the other single strand molecule is a naturally occurring nucleic acid, e.g., a genomic molecule, or chromosome, e.g., a viral, bacterial, plant, or animal nucleic acid, one single strand molecule is a probe and one single strand molecule is the target sequence to be detected or amplified in an amplification reaction, or in a reaction in which amplification or detection is based on the amplification or detection of the probe or a fragment of the probe
  • the method further including detecting a product ofthe reactions
  • the invention features methods of performing a cycling reaction between a single strand binding ligand formed from a duplex nucleic acid molecule comprising a first (e.g., a probe) and a second (e.g., a target) single strand nucleic acid molecule comprising: (1 ) forming a reaction mixture comprising a plurality of the first single strand molecules, at least one of the second single strand molecules, a single strand binding ligand, and a duplex strand binding ligand, an amount, concentration, or chemical potential of the single strand binding ligand and an amount, concentration, or chemical potential ofthe duplex strand binding ligand being such that the following cycle of events can occur under isothermal conditions,
  • n is at least 1 , 2, 5, 10, 25, 50, 100,
  • Such methods may be applied to a wide variety of cycling or catalytic reactions, as discussed above.
  • the single strand binding ligand is a purified natural product or a non-naturally occurring product DEFINE
  • the concentration, number of molecules present, or chemical potential of the single strand binding ligand is greater than the concentration, number of molecules present, or chemical potential of the first single strand, the second single strand, both the first single strand and the second single strand, the duplex, or the combination of the first single strand, the second single strand, and the duplex, e.g., at least n-fold greater, wherein n is at least 1 , 2, 5, 10, 25, 50, 100, 500, 10 3 , 10 4 , 10 5 , or 10 6 .
  • the single strand binding ligand comprises any of: a compound which binds to a single strand nucleic acid in a sequence- specific way; a compound which binds to a single strand nucleic acid in a sequence-non- specific way; a protein; an enzyme; an enzyme which alters the structure of a nucleic acid to which it binds; an enzyme which alters the structure of a nucleic acid to which it binds by breaking or forming a covalent or non-covalent bond, between an atom of the nucleic acid and another atom, an enzyme which cleaves a nucleic acid to which it binds, a restriction enzyme, a restriction endonuclease, an enzyme which methylates the nucleic acid to which it binds; an enzyme which alkylates the duplex nucleic acid to which it binds, an enzyme which alters the primary or secondary structure of a duplex nucleic acid to which it binds; an enzyme which promotes
  • the concentration, number of molecules present, or chemical potential of the single strand binding ligand is no more than 10 times the concentration, number of molecules present, or chemical potential of the duplex-binding ligand; the concentration, number of molecules present, or chemical potential of the single strand binding ligand is no less than one-tenth the concentration, number of molecules present, or chemical potential of the duplex-binding ligand, the concentration, number of molecules present, or chemical potential of the duplex-binding ligand is no more than 10 times the concentration, number of molecules present, or chemical potential of the single strand binding ligand, the concentration, strand in the highest concentration is greater than 1 1, 2 1, 5 1 , 10 1 , 25 1 , 50 1, 100 1 , or 10 n 1, wherein n is an integer between 3 and 10, inclusive
  • the single strand binding ligand comprises any of a compound which binds to a single strand nucleic acid in a sequence- specific way, a compound which binds to a single strand nucleic acid in
  • one or both single strand molecules are DNA; one or both single strand molecules are RNA; one single strand molecule is RNA and the other is DNA, one single strand molecule is a single stranded probe and the other single strand molecule is a target sequence which is to be cleared, detected, or amplified, one single stand molecule is a synthetic, purified natural, genetically engineered, or recombinant DNA or RNA molecule, and the other single strand molecule is a naturally occurring nucleic acid, e.g., a genomic molecule, or chromosome, e.g., a viral, bacterial, plant, or animal nucleic acid, one single strand molecule is a probe and one single strand molecule is the target sequence to be detected or amplified in an amplification reaction, or in a reaction in which amplification or detection is based on the amplification or detection ofthe probe or a fragment ofthe probe
  • the invention features methods of cyclically reacting a single strand binding ligand with single strand formed from a duplex formed by a hybridization of a first single strand molecule (probe) to a second single strand molecule (target) including forming a reaction mixture comprising the first single strand molecule, the second single strand molecule, the single strand binding ligand, and a duplex-binding ligand, the chemical potential of the duplex-binding ligand being sufficient to cause the formation of duplex, and the chemical potential of the single strand binding ligand being such that duplex dissociation reaction occurs concurrently with the formation reaction, the chemical potentials being such that the rate of duplex formation and the rate of duplex dissociation are such that a cycle of (duplex dissociation)-(single strand binding ligand:single strand binding)-(duplex formation) could occur under isothermal conditions, and allowing the cycle to proceed
  • Such methods may be applied to a wide variety of cycling or catalytic
  • the single strand binding ligand comprises any of a compound which binds to a single strand nucleic acid in a sequence- specific way, a compound which binds to a single strand nucleic acid in a sequence non specific way, a protein, an enzyme, an enzyme which alters the structure ofa nucleic acid to which it binds, an enzyme which alters the structure of a nucleic acid to which it binds by breaking or forming a covalent non-covalent bond, between an atom of the nucleic acid and another atom, an enzyme which cleaves a nucleic acid to which it binds, a restriction enzyme, a restriction endonuclease, an enzyme which methylates the nucleic acid to which it binds; an enzyme which alkylates the duplex nucleic acid to which it binds, an enzyme which alters the primary or secondary structure of a duplex nucleic acid to which it binds, an enzyme which promotes or inhibits recombin
  • the concentration, number of molecules present, or chemical potential of the single strand binding ligand is no more than 10 times the concentration, number of molecules present, or chemical potential of the duplex-binding ligand, the concentration, number of molecules present, or chemical potential of the single strand binding ligand is no less than one-tenth the concentration, number of molecules present, or chemical potential of the duplex-binding ligand, the concentration, number of molecules present, or chemical potential of the duplex-binding ligand is no more than 10 times the concentration, number of molecules present, or chemical potential of the single strand binding ligand, the concentration, number of molecules present, or chemical potential of the duplex-binding ligand is no less than one- tenth the concentration, number of molecules present, or chemical potential of the single strand binding ligand
  • the concentration, number of molecules present, or chemical potential of the duplex-binding ligand and the concentration, number of molecules present, or chemical potential ofthe single strand binding ligand are such that the rate of formation of duplex is substantially equal to the rate of formation of single strands
  • the concentration, number of molecules present, or chemical potential of the duplex-binding ligand and the concentration, number of molecules present, or chemical potential of the single strand binding ligand are such that the rate of formation of duplex is no more than 2, 5, 10, 20, 50, 100, 10 3 or 10 4 times the rate of formation of single strands
  • the concentration, number of molecules present, or chemical potential of the duplex-binding ligand and the concentration, number of molecules present, or chemical potential of the single strand binding ligand are such that the rate of formation of single strands is no more than 2, 5, 10, 20, 50, 100, 10 J or 10 4 times the rate of formation of duplex
  • the method is performed under isothermal conditions; the temperature is below the Tm ofthe duplex, the temperature is at least n C°, wherein n is 5, 10, 20, 30, 40, 50, 60, 70, or 80, below the Tm, at least one of the strand molecules is a purified nucleic acid molecule, the number of molecules of, or the chemical potential of, the duplex-binding ligand is greater than the concentration, number of molecules present, or chemical potential of the first single strand, the second single strand, both the first single strand and the second single strand, the duplex, or the combination of the first single strand, the second single strand, and the duplex, e.g., at least n-fold greater, wherein n is at least 1 , 2, 5, 10, 25, 50, 100, 500, 10 3 , 10 4 , 10 5 , or 10 6 ; the first single strand molecule and the second single strand molecule are present in substantially equal amounts or at substantially equal chemical potentials in the reaction mix, the first single
  • DNA, one or both single strand molecules are RNA, one single strand molecule is RNA and the other is DNA, one single strand molecule is a single stranded probe and the other single strand molecule is a target sequence which is to be cleared, detected, or amplified, one single strand molecule is a synthetic, purified natural, genetically engineered, or recombinant DNA or RNA molecule, and the other single strand molecule is a naturally occurring nucleic acid, e.g., a genomic molecule, or chromosome, e.g., a viral, bacterial, plant, or animal nucleic acid, one single strand molecule is a probe and one single strand molecule is the target sequence to be detected or amplified in an amplification reaction, or in a reaction in which amplification or detection is based on the amplification or detection ofthe probe or a fragment of the probe
  • the invention features a reaction mixture comprising a single strand nucleic acid molecule (e.g., a probe, as discussed above), a second single strand nucleic acid molecule, a duplex and a duplex-binding ligand as discussed above at an amount, concentration, or chemical potential which results in the formation of a duplex at a rate which is substantially greater than the rate in the absence of the duplex- binding ligand
  • the duplex-binding ligand is present at an amount, concentration, or chemical potential sufficient that the rate of duplex formation is increased by at least n-fold, wherein n is 2, 5, 10, 50, 100, 500, 10 3 , 10 4 , 10-5, ] )6, by the addition of duplex-binding ligand, the rate of duplex formation in the absence of duplex-binding ligand is substantially zero, at least one of the strand single molecules is a purified nucleic acid molecule, the duplex-binding ligand is a purified natural product or a product which does not naturally occur in the living cells
  • the concentration, number of molecules of, or the chemical potential of, the duplex-binding ligand is greater than the concentration, number of molecules present, or chemical potential of the first single strand, the second single strand, both the first single strand and the second single strand, the duplex, or the combination ofthe first single strand, the second single strand, and the duplex, e.g
  • the first single strand molecule and the second single strand molecule are present in substantially equal amounts or at substantially equal concentration, number, or chemical potentials in the reaction mix, the first single strand molecule is a probe or primer molecule, the second single strand molecule is a target molecule, the first single strand molecule is a probe or primer molecule, the second single strand molecule is a target molecule and the ratio by weight, molarity, number, concentration, or chemical potential of the first sequence to the second sequence is less than or equal to 1 1 , 2 1 , 5 1 , 10 1 , 25 1 , 50 1, 100 1 , or 10 n 1, wherein n is an integer between 3 and 10, inclusive, the duplex-binding ligand is present at an amount, concentration, or chemical potential sufficient that wherein the free energy for the formation of the duplex from the first and the second sequence is decreased by at least n-fold, wherein n is 2, 5, 10, 50, 100, 500, 1 ,000, by addition of the duplex-
  • the duplex-binding ligand comprises any of a compound which binds to a duplex nucleic acid in a sequence-specific way, a compound which binds to a duplex nucleic acid in a sequence-non-specific way, a protein, an enzyme, an enzyme which alters the structure of a duplex nucleic acid to which is binds, an enzyme which alters the structure of a duplex nucleic acid to which it binds by breaking or forming a covalent or non-covalent bond, between an atom of the nucleic acid and another atom, an enzyme which cleaves one or both strands of a duplex nucleic acid to which it binds, a restriction enzyme, a restriction endonuclease, an enzyme which methylates the duplex to which it binds, an enzyme which alleviates the duplex nucleic acid to which it binds, a nucleic acid ligase, e.g.
  • DNA ligase an enzyme which promotes or catalyzes the synthesis of a nucleic acid, a nucleic acid polymerase, a nucleic acid polymerase which requires a double stranded primer, a DNA polymerase, DNA polymerase 1, Taq polymerase, an RNA polymerase, an enzyme which alters the primary or secondary structure of a duplex nucleic acid to which it binds, a topoisomerase, an enzyme which promotes or inhibits recombination, a DNA binding ligand, a mutagen, a compound which enhances the expression of a gene under the control of the duplex bound by a ligand, a compound which intercalates into a double stranded nucleic acid, a compound which, when contacted with a reaction mixture comprising a first single stranded nucleic acid and a second single stranded nucleic acid will accelerate the rate of duplex formation at least n-fold, wherein n is 2, 5,
  • DNA, one or both single strand molecules are RNA, one single strand molecule is RNA and the other is DNA, one single strand molecule is a single stranded probe and the other single strand molecule is a target sequence which is to be cleared, detected, or amplified, one single strand molecule is a synthetic, purified natural, genetically engineered, or recombinant DNA or RNA molecule, and the other single strand molecule is a naturally occurring nucleic acid, e.g., a genomic molecule, or chromosome, e.g., a viral, bacterial, plant, or animal nucleic acid; one single strand molecule is a probe and one single strand molecule is the target sequence to be detected or amplified in an amplification reaction, or in a reaction in which amplification or detection is based on the amplification or detection ofthe probe or a fragment ofthe probe
  • the duplex-binding ligand is a polymerase, e.g., a DNA polymerase, or a ligase
  • the invention features a reaction mixture including a duplex-binding ligand, a first single strand nucleic acid molecule, a second single strand nucleic acid molecule, the duplex-binding ligand being present at an amount, concentration, or chemical potential which results in the formation of a duplex at a temperature substantially above the Tm ofthe duplex ofthe first and second strands
  • the temperature is at least n C°, wherein n is 5, 10, 20, 30, 40, 50, 60, 70, or 80, above the Tm, the duplex-binding ligand is present at a chemical potential sufficient that wherein the rate of duplex formation is increased by at least n-fold, wherein n is 2, 5, 10, 50, 100, 500, 10 3 , 10 4 , 10 5 , 10 6 , by the addition of duplex-binding ligand, the rate of duplex formation in the absence of duplex-binding ligand is substantially zero, at least one of the strand molecules is a purified nucleic acid molecule, the duplex-binding ligand is a purified natural product or a product which does not naturally occur in the living cells
  • the concentration, number of molecules of, or the chemical potential of, the duplex-binding ligand is greater than the concentration, number of molecules present, or chemical potential of the first single strand, the second single strand, both the first single strand and the second single strand, the duplex, or the combination ofthe first single strand, the second single strand, and the duplex, e.g., at least n-fold greater, wherein n is at least 1 , 2, 5, 10, 25, 50, 100, 500, 10 3 , 10 4 , 10 5 , or 10 6
  • the first single strand molecule and the second single strand molecule are present in substantially equal amounts or at substantially equal concentration, number, or chemical potentials in the reaction mix, the first single strand molecule is a probe or primer molecule, the second single strand molecule is a target molecule, the first single strand molecule is a probe or primer molecule, the second single strand molecule is a target molecule and the ratio by weight, molarity, number, concentration, or chemical potential of the first sequence to the second sequence is less than or equal to 1 1 , 2 1 , 5 1, 10 1 , 25 1 , 50 1 , 100 1, or 10 n 1, wherein n is an integer between 3 and 10, inclusive, the duplex-binding ligand is present at an amount, concentration, or chemical potential sufficient that wherein the free energy for the formation of the duplex from the first and the second sequence is decreased by at least n-fold, wherein n is 2, 5, 10, 50, 100, 500, 1 ,000, by addition of the duplex-binding
  • the duplex-binding ligand comprises any of a compound which binds to a duplex nucleic acid in a sequence-specific way, a compound which binds to a duplex nucleic acid in a sequence-non-specific way, a protein, an enzyme, an enzyme which alters the structure of a duplex nucleic acid to which is binds, an enzyme which alters the structure of a duplex nucleic acid to which it binds by breaking or forming a covalent or non-covalent bond, between an atom of the nucleic acid and another atom, an enzyme which cleaves one or both strands of a duplex nucleic acid to which it binds, a restriction enzyme, a restriction endonuclease, an enzyme which methylates the duplex to which it binds, an enzyme which alkylates the duplex nucleic acid to which it binds, a nucleic acid ligase, e.g., DNA ligase,
  • DNA, one or both single strand molecules are RNA, one single strand molecule is RNA and the other is DNA, one single strand molecule is a single stranded probe and the other single strand molecule is a target sequence which is to be cleared, detected, or amplified, one single strand molecule is a synthetic, purified natural, genetically engineered, or recombinant DNA or RNA molecule, and the other single strand molecule is a naturally occurring nucleic acid, e.g., a genomic molecule, or chromosome, e.g., a viral, bacterial, plant, or animal nucleic acid, one single strand molecule is a probe and one single strand molecule is the target sequence to be detected or amplified in an amplification reaction, or in a reaction in which amplification or detection is based on the amplification or detection ofthe probe or a fragment of the probe
  • the invention features reaction mixtures comprising a plurality of first single strand molecules (e.g., probes as discussed above), at least one second single strand molecule (e.g., a target), a single strand binding ligand, and a duplex strand binding ligand (as discussed above), an amount, concentration, or chemical potential of the single strand binding ligand and the chemical potential of the duplex strand binding ligand being such that the following cycle of events can occur under isothermal conditions,
  • the cycle can occur at least n times, wherein n is at least 1, 2, 5, 10, 25, 50, 100, 500, 10 3 , 10 4 , 10 5 , or 10 ⁇
  • the single strand binding ligand is a purified natural product or a non-naturally occurring product DEFINE, the concentration, number of molecules present, or chemical potential of the single strand binding ligand is greater than the concentration, number of molecules present, or chemical potential of the first single strand, the second single strand, both the first single strand and the second single strand, the duplex, or the combination of the first single strand, the second single strand, and the duplex, e.g., at least n-fold greater, wherein n is at least 1, 2, 5, 10, 25, 50, 100, 500, 10 3 , 10 4 , 10 5 , or 10 6
  • the single strand binding ligand comprises any of a compound which binds to a single strand nucleic acid in a sequence- specific way, a compound which binds to a single strand nucleic acid in a sequence-non ⁇ specific way, a protein, an enzyme, an enzyme which alters the structure of a nucleic acid to which it binds, an enzyme which alters the structure of a nucleic acid to which it binds by breaking or forming a covalent or non-covalent bond, between an atom of the nucleic acid and another atom, an enzyme which cleaves a nucleic acid to which it binds, a restriction enzyme, a restriction endonuclease, an enzyme which methylates the nucleic acid to which it binds, an enzyme which alkylates the duplex nucleic acid to which it binds, an enzyme which alters the primary or secondary structure of a duplex nucleic acid to which it binds, an enzyme which promotes or inhibit
  • the concentration, number of molecules present, or chemical potential of the single strand binding ligand is no more than 10 times the concentration, number of molecules present, or chemical potential of the duplex-binding ligand; the concentration, number of molecules present, or chemical potential of the single stand binding ligand is no less than one-tenth the concentration, number of molecules present, or chemical potential of the duplex-binding ligand; the concentration, number of molecules present, or chemical potential of the duplex-binding ligand is no more than 10 times the concentration, number of molecules present, or chemical potential of the single strand binding ligand, the concentration, number of molecules present, or chemical potential of the duplex-binding ligand is no less than one- tenth the concentration, number of molecules present, or chemical potential of the single strand binding ligand
  • the temperature is above the Tm; the temperature is at least n C°
  • the duplex-binding ligand comprises any of a compound which binds to a duplex nucleic acid in a sequence-specific way, a compound which binds to a duplex nucleic acid in a sequence-non-specific way; a protein, an enzyme, an enzyme which alters the structure of a duplex nucleic acid to which is binds, an enzyme which alters the structure of a duplex nucleic acid to which it binds by breaking or forming a covalent or non-covalent bond, between an atom of the nucleic acid and another atom, an enzyme which cleaves one or both strands of a duplex nucleic acid to which it binds, a restriction enzyme, a restriction endonuclease, an enzyme which methylates the duplex to which it binds, an enzyme which alkylates the duplex nucleic acid to which it binds, a nucleic acid ligase, e.g., DNA ligase,
  • the duplex-binding ligand is a polymerase, e.g., a DNA polymerase, or a ligase; the single strand binding ligand is present in sufficient concentration, number of molecules, or an amount, concentration or chemical potential such that there is substantially no non-specific hybridization of the first single strand to a sequence other than the second single strand
  • a reaction mixture including a plurality of the first single strand molecules, at least one second single strand molecule, a single strand binding ligand, and a duplex-binding ligand comprising polymerase, an amount, concentration, or chemical potential of the single strand binding ligand and an amount, concentration, or chemical potential of the duplex-binding ligand being such that the following cycle of events can occur under isothermal conditions,
  • the cycle occurs at least n times, wherein n is at least 1, 2, 5, 10, 25, 50, 100, 500, 10 3 , 10 4 , 10 5 , or 10 6 ;
  • the single strand binding ligand is a purified natural product or a non-naturally occurring product; the concentration, number of molecules present, or chemical potential of the single strand binding ligand is greater than the concentration, number of molecules present, or chemical potential ofthe first single strand, the second single strand, both the first single strand and the second single strand, the duplex, or the combination of the first single strand, the second single strand, and the duplex, e.g., at least n-fold greater, wherein n is at least 1, 2, 5, 10, 25, 50, 100, 500, 10 3 , 10 4 , 10 5 , or 10 6
  • the single strand binding ligand comprises any of a compound which binds to a single strand nucleic acid in a sequence- specific way; a compound which binds to
  • the concentration, number of molecules present, or chemical potential of the single strand binding ligand is no more than 10 times the concentration, number of molecules present, or chemical potential of the duplex-binding ligand; the concentration, number of molecules present, or chemical potential of the single strand binding ligand is no less than one-tenth the concentration, number of molecules present, or chemical potential of the duplex-binding ligand; the concentration, number of molecules present, or chemical potential of the duplex -binding ligand is no more than 10 times the concentration, number of molecules present, or chemical potential of the single strand binding ligand, the concentration, number of molecules present, or chemical potential of the duplex-binding ligand is no less than one- tenth the concentration, number of molecules present, or chemical potential of the single stand binding ligand In other preferred embodiments the concentration, number of molecules present, or chemical potential of the duplex-binding ligand and the concentration, number of molecules present, or chemical potential ofthe single strand binding ligand are such that the rate of formation
  • the temperature is above the Tm, the temperature is at least n C°, wherein n is 5, 10, 20, 30, 40, 50, 60, 70, or 80, above the Tm; at least one ofthe single strand molecules is a purified nucleic acid molecule
  • the number of molecules of, or the chemical potential of, the duplex-binding ligand is greater than the concentration, number of molecules present, or chemical potential of the first single strand, the second single strand, both the first single strand and the second single strand, the duplex, or the combination of the first single strand, the second single strand, and the duplex, e.g., at least n-fold greater, wherein n is at east 1 , 2, 5, 10, 25, 50, 100, 500, 10 3 , 10 4 , 10 5 , or 10 6
  • the first single strand molecule and the second single strand molecule are present in substantially equal amounts or at substantially equal an amount, concentration, or chemical potentials in the reaction mix, the first single strand molecule is a probe or primer molecule, the second single strand molecule is a target molecule, the first single strand molecule is a probe or primer molecule, the second single strand molecule is a target molecule and the ratio by weight, molarity, number, concentration, or chemical potentials of the first sequence to the second sequence is less than or equal to 1 1 , 2 1, 5 1 , 10 1 , 25 1 , 50 1, 100.1, or 10 n 1, wherein n is an integer between 3 and 10, inclusive, the ratio by weight, molarity, number, concentration, or chemical potentials of the duplex-binding ligand to the single strand in the highest concentration is greater than 1 1 , 2 1 , 5 1 , 10 1, 25 1 , 50 1, 100 1, or 10 n 1, wherein n is an integer between 3 and 10,
  • the duplex -binding ligand in addition to a polymerase, comprises any of a compound which binds to a duplex nucleic acid in a sequence-specific way, a compound which binds to a duplex nucleic acid in a sequence- non-specific way, a protein, an enzyme, an enzyme which alters the structure of a duplex nucleic acid to which is binds, an enzyme which alters the structure of a duplex nucleic acid to which it binds by breaking or forming a covalent or non-covalent bond, between an atom of the nucleic acid and another atom, an enzyme which cleaves one or both strands of a duplex nucleic acid to which it binds, a restriction enzyme, a restriction endonuclease, an enzyme which methylates the duplex to which it binds, an enzyme which alkylates the duplex nucleic acid to which it binds, a nucleic acid ligase, e
  • one or both single strand molecules are DNA; one or both single strand molecules are RNA; one single strand molecule is RNA and the other is DNA; one single strand molecule is a single stranded probe and the other single strand molecule is a target sequence which is to be cleared, detected, or amplified, one single strand molecule is a synthetic, purified natural, genetically engineered, or recombinant DNA or RNA molecule, and the other single strand molecule is a naturally occurring nucleic acid, e.g., a genomic molecule, or chromosome, e.g., a viral, bacterial, plant, or animal nucleic acid, one single strand molecule is a probe and one single strand molecule is the target sequence to be detected or amplified in an amplification reaction, or in a reaction in which amplification or detection is based on the amplification or detection ofthe probe or a fragment of the probe
  • the single strand binding ligand is present in sufficient concentration, number of molecules, or chemical potential such that there is substantially no non-specific hybridization of the first single strand to a sequence other than the second single strand
  • the invention features reaction mixtures including a duplex -binding ligand (as discussed above), a first single strand molecule (e.g., a probe, as discussed above), a second single strand molecule, and a single strand binding ligand, the chemical potential of the duplex-binding ligand being sufficient to cause the formation of duplex, and the chemical potential of the single strand binding ligand being such that duplex dissociation reaction occurs concurrently with the formation reaction, the chemical potentials being such that the rate of duplex formation and the rate of duplex dissociation are such that a cycle of (duplex-binding ligand duplex-binding)- (duplex dissociation)-(duplex-binding ligand:duplex-binding) could occur under isothermal conditions
  • the cycle can occur least n times under isothermal conditions, wherein n is at least 1 , 2, 5, 10, 25, 50, 100, 500, 10 3 , 10 4 , 10 5 , or 10 * -5;
  • the single strand binding ligand is a purified natural product or a non-naturally occurring product;
  • the concentration, number of molecules present, or chemical potential ofthe single strand binding ligand is greater than the concentration, number of molecules present, or chemical potential of the first single strand, the second single strand, both the first single strand and the second single strand, the duplex, or the combination ofthe first single strand, the second single strand, and the duplex, e.g., at least n-fold greater, wherein n is at least 1 , 2, 5, 10, 25, 50, 100, 500, 10 3 , 10 4 , 10 5 , or 10 ⁇ 5
  • the single strand binding ligand includes any of. a compound which binds to a single strand nucleic acid in a sequence-specific way; a compound which binds to a single strand nucleic acid in a sequence-nonspecific way, a protein, an enzyme; an enzyme which alters the structure of a nucleic acid to which it binds, an enzyme which alters the structure of a nucleic acid to which it binds by breaking or forming a covalent or non-covalent bond, between an atom of the nucleic acid and another atom, an enzyme which cleaves a nucleic acid to which it binds, a restriction enzyme; a restriction endonuclease; an enzyme which methylates the nucleic acid to which it binds, an enzyme which alkylates the duplex nucleic acid to which it binds; an enzyme which alters the primary or secondary structure of a duplex nucleic acid to which it binds; an enzyme which promotes or
  • the temperature is above the Tm; the temperature is at least n C°, wherein n is 5, 10, 20, 30, 40, 50, 60, 70, or 80, above the Tm; at least one ofthe strand molecules is a purified nucleic acid molecule
  • the number of molecules of, or the chemical potential of, the duplex-binding ligand is greater than the concentration, number of molecules present, or chemical potential of the first single strand, the second single strand, both the first single strand and the second single strand, the duplex, or the combination of the first single strand, the second single strand, and the duplex, e.g., at least n-fold greater, wherein n is at least 1, 2, 5, 10, 25, 50, 100, 500, 10 3 , 10 4 , 10 5 or 10 6
  • the first single strand molecule and the single strand molecule are present in substantially equal amounts or at substantially equal chemical potentials in the reaction mix, the first single strand molecule is a probe or primer molecule, the second single strand molecule is a target molecule, the first single strand molecule is a probe or primer molecule, the second single strand molecule is a target molecule and the ratio by weight, molarity, number, concentration, or chemical potentials of the first sequence
  • the duplex-binding ligand comprises any of a compound which binds to a duplex nucleic acid in a sequence-specific way, a compound which binds to a duplex nucleic acid in a sequence-non-specific way, a protein, an enzyme, an enzyme which alters the structure of a duplex nucleic acid to which is binds, an enzyme which alters the structure of a duplex nucleic acid to which it binds by breaking or forming a covalent or non-covalent bond, between an atom of the nucleic acid and another atom, an enzyme which cleaves one or both strands of a duplex nucleic acid to which it binds, a restriction enzyme, a restriction endonuclease, an enzyme which methylates the duplex to which it binds, an enzyme which alkylates the duplex nucleic acid to which it binds, a nucleic acid ligase, e.g., DNA ligase,
  • the duplex-binding ligand is a polymerase, e.g., a DNA polymerase, or a ligase
  • the single strand binding ligand is present in sufficient concentration, number of molecules, or chemical potential such that there is substantially no non-specific hybridization of the first single strand to a sequence other than the second single strand
  • the invention features reaction mixtures including a first single strand molecule, a second single strand molecule, a single strand binding ligand, and a duplex-binding ligand comprising a polymerase, a ligase, or other sequence forming enzyme, the chemical potential of the duplex-binding ligand being sufficient to cause the formation of duplex, and the chemical potential of the single strand binding ligand being such that a predetermined level of duplex dissociation reaction occurs, the rate of duplex formation and the rate of duplex dissociation being such that a cycle of duplex-binding ligand catalyzed formation-duplex dissociation-duplex-binding ligand catalyzed formation which can occur under isothermal conditions.
  • the single strand binding ligand is a purified natural product or a non-naturally occurring product, the concentration, number of molecules present, or chemical potential of the single strand binding ligand is greater than the concentration, number of molecules present, or chemical potential of the first single strand, the second single strand, both the first single strand and the second single strand, the duplex, or the combination ofthe first single strand, the second single strand, and the duplex, e.g., at least n-fold greater, wherein n is at least 1 , 2, 5, 10, 25, 50, 100, 500, 10 3 , 10 4 , 10 5 , or 10 6
  • the single strand binding ligand comprises any of a compound which binds to a single strand nucleic acid in a sequence- specific way, a compound which binds to a single strand nucleic acid in a sequence-non ⁇ specific way; a protein, an enzyme, an enzyme which alters the structure of a nucleic acid to
  • the concentration, number of molecules present, or chemical potential of the single strand binding ligand is no more than 10 times the concentration, number of molecules present, or chemical potential of the duplex-binding ligand; the concentration, number of molecules present, or chemical potential of the single strand binding ligand is no less than one-tenth the concentration, number of molecules present, or chemical potential of the duplex-binding ligand, the concentration, number of molecules present, or chemical potential of the duplex-binding ligand is no more than 10 times the concentration, number of molecules present, or chemical potential of the single strand binding ligand, the concentration, number of molecules present, or chemical potential of the duplex-binding ligand is no less than one- tenth the concentration, number of molecules present, or chemical potential of the single strand binding ligand
  • the concentration, number of molecules present, or chemical potential of the duplex-binding ligand and the concentration, number of molecules present, or chemical potential ofthe single strand binding ligand are such that the rate of formation of duplex is substantially equal to the rate of formation of single strands
  • the concentration, number of molecules present, or chemical potential of the duplex-binding ligand and the concentration, numbei of molecules present, or chemical potential of the single strand binding ligand are such that the rate of formation of duplex is no more than 2, 5, 10, 20, 50, 100, 10 J or 10 4 times the rate of formation of single strands
  • the concentration, number of molecules present, or chemical potential of the duplex-binding ligand and the concentration, number of molecules present, or chemical potential of the single strand binding ligand are such that the rate of formation of single strands is no more than 2, 5, 10, 20, 50, 100, 10 3 or 10 4 times the rate of formation of duplex
  • the cycle can occur above the Tm of the duplex; the cycle can occur at least n C° above the Tm of the duplex, wherein n is 5, 10, 20, 30, 40, 50, 60, 70, or 80, at least one of the strand molecules is a purified nucleic acid molecule, the number of molecules of, or the chemical potential of, the duplex- binding ligand is greater than the concentration, number of molecules present, or chemical potential of the first single strand, the second single strand, both the first single strand and the second single strand, the duplex, or the combination of the first single strand, the second single strand, and the duplex, e.g , at least n-fold greater, wherein n is at least 1, 2, 5, 10, 25, 50, 100, 500, 10 3 , 10 4 , I 0 5 , or 10 6 , the first single strand molecule and the second single strand molecule are present in substantially equal amounts or at substantially equal an amount, concentration, or chemical potentials in the reaction mix, the first single single strand
  • the first single strand molecule is a probe or primer molecule
  • the second single strand molecule is a target molecule and the ratio by weight, molarity, number, concentration, or chemical potentials of the first sequence to the second sequence is less than or equal to 1 I , 2 1, 5 1, 10 1, 25 1, 50 1, 100 1, or 10 n 1 , wherein n is an integer between 3 and 10, inclusive
  • the ratio by weight, molarity, number, concentration, or chemical potentials ofthe duplex-binding ligand to the single strand in the highest concentration is greater than 1 1, 2 1, 5 1, 10 1 , 25 1 , 50 1 , 100 1 , or 10 n 1, wherein n is an integer between 3 and 10, inclusive
  • the duplex-binding ligand comprises any of a compound which binds to a duplex nucleic acid in a sequence-specific way, a compound which binds to a duplex nucleic acid in a sequence-non-specific way, a protein, an enzyme, an enzyme which alters the structure of a duplex nucleic acid to which it binds, an enzyme which alters the structure of a duplex nucleic acid to which it binds by breaking or forming a covalent or non-covalent bond, between an atom of the nucleic acid and another atom, an enzyme which cleaves one or both strands of a duplex nucleic
  • one or both single strand molecules are DNA, one or both single strand molecules are RNA, one single strand molecule is RNA and the other is DNA, one single strand molecule is a single stranded probe and the other single strand molecule is a target sequence which is to be cleared, detected, or amplified, one single strand molecule is a synthetic, purified natural, genetically engineered, or recombinant DNA or RNA molecule, and the other single strand molecule is a naturally occurring nucleic acid, e.g., a genomic molecule, or chromosome, e.g., a viral, bacterial, plant, or animal nucleic acid, one single strand molecule is a probe and one single strand molecule is the target sequence to be detected or amplified in an amplification reaction, or in a reaction in which amplification or detection is based on the amplification or detection ofthe probe or a fragment of the probe
  • the single strand binding ligand is present in sufficient concentration, number of molecules, or chemical potential such that there is substantially no non-specific hybridization of the first single strand to a sequence other than the second single strand
  • the invention features reaction mixtures including a single strand binding ligand, a first single strand nucleic acid molecule (e.g., a probe, as discussed above), a second single strand nucleic acid molecule, and a duplex-binding ligand (as discussed above), the single strand binding ligand being present at a chemical potential which results in the formation of a single strand at a rate which is substantially greater than the rate in the absence of the single strand binding ligand, thereby allowing the reaction between the single strand and the single strand binding ligand to proceed
  • the rate of single strand formation is increased by at least n-fold, wherein n is 2, 5, 10, 50, 100, 500, 10 3 , 10 4 , 10 5 , 10 6 , by the addition of single strand binding ligand, the rate of single strand formation in the absence of single strand binding ligand is substantially zero, at least one of the single strand molecules is a purified nucleic acid molecule, the single strand binding ligand is a purified natural product or a product which does not naturally occur in the living cells
  • the concentration, number of molecules of, or the chemical potential of, the single strand binding ligand is greater than the concentration, number of molecules present, or chemical potential of the first single strand, the second single strand, both the first single strand and the second single strand, the duplex, or the combination ofthe first single strand, the second single strand, and the duplex, e.g., at least n-fold greater, wherein n is at least 1, 2, 5, 10, 25, 50, 100, 500, 10 3 , 10 4 , 10 5 , or l ⁇ 6
  • first single strand molecule and the second single strand molecule are present in substantially equal amounts or at substantially equal concentration, number, or chemical potentials in the reaction mix
  • the first single strand molecule is a probe or primer molecule
  • the second single strand molecule is a target molecule
  • the first single strand molecule is a probe or primer molecule
  • the second single strand molecule is a target molecule
  • the ratio by weight, molarity, number, concentration, or chemical potential ofthe first sequence to the second sequence is less than or equal to 1 1, 2 1, 5 1 , 10 1, 25 1, 50 1, 100 1 , or 10 n 1 , wherein n is an integer between 3 and 10, inclusive, the free energy for the formation of the single strand from the duplex is decreased by at least n-fold, wherein n is 2, 5, 10, 50, 100, 500, 1,000, by addition of the single strand binding ligand to the reaction mix, the ratio by weight, molarity, number, concentration, or chemical potential of the single strand binding ligand to the single strand in the highest concentration is greater than 1 1, 2 1, 5 1, 10 1, 25 1, 50 1, 100 1, or 10 n 1, wherein n is an integer
  • the single strand binding ligand comprises any of a compound which binds to a single strand nucleic acid in a sequence- specific way, a compound which binds to a single strand nucleic acid in a sequence-non ⁇ specific way, a protein, an enzyme, an enzyme which alters the structure of a nucleic acid to which is binds, an enzyme which alters the structure of a nucleic acid to which it binds by breaking or forming a covalent or non-covalent bond, between an atom of the nucleic acid and another atom; an enzyme which cleaves a nucleic acid to which it binds, an enzyme which alters the primary or secondary structure of a nucleic acid to which it binds; a topoisomerase; an enzyme which promotes or inhibits recombination; a DNA binding ligand; a mutagen, a compound which enhances the expression of a gene under the control of the nucleic acid bound by the ligand;
  • one or both single strand molecules are DNA; one or both single strand molecules are RNA; one single strand molecule is RNA and the other is DNA; one single strand molecule is a single stranded probe and the other single strand molecule is a target sequence which is to be cleared, detected, or amplified, one single strand molecule is a synthetic, purified natural, genetically engineered, or recombinant DNA or RNA molecule, and the other single strand molecule is a naturally occurring nucleic acid, e.g., a genomic molecule, or chromosome, e.g., a viral, bacterial, plant, or animal nucleic acid; one single strand molecule is a probe and one single strand molecule is the target sequence to be detected or amplified in an amplification reaction, or in a reaction in which amplification or detection is based on the amplification or detection ofthe probe or a fragment ofthe probe.
  • a naturally occurring nucleic acid e.g., a
  • the invention features reaction mixtures including: a first single strand nucleic acid molecule (e.g., a probe, as discussed above); a second single strand nucleic acid molecule, and a single strand binding ligand, the single strand binding ligand being present at a chemical potential which results in the formation of a single strand at a temperature substantially less than the Tm of the duplex.
  • a first single strand nucleic acid molecule e.g., a probe, as discussed above
  • a second single strand nucleic acid molecule e.g., a probe, as discussed above
  • a single strand binding ligand e.g., the single strand binding ligand
  • the temperature is at least n C°, wherein n is 5, 10, 20, 30, 40, 50, 60, 70, or 80, below the Tm; the rate of duplex formation is increased by at least n-fold, wherein n is 2, 5, 10, 50, 100, 500, 10 3 , 10 4 , 10 5 , 10 6 , by the addition of duplex-binding ligand; the rate of single strand formation in the absence of single strand binding ligand is substantially zero; at least one ofthe strand molecules is a purified nucleic acid molecule; the single strand binding ligand is a purified natural product or a product which does not naturally occur in the living cells.
  • the concentration, number of molecules of, or the chemical potential of, the single strand binding ligand is greater than the concentration, number of molecules present, or chemical potential of the first single strand, the second single strand, both the first single strand and the second single strand, the duplex, or the combination ofthe first single strand, the second single strand, and the duplex, e.g., at least n-fold greater, wherein n is at least 1, 2, 5, 10, 25, 50, 100, 500, 10 3 , 10 4 , 10 5 , or 10 6 ; the first single strand molecule and the second single strand molecule are present in substantially equal amounts or at substantially equal concentration, number, or chemical potentials in the reaction mix, the first single strand molecule is a probe or primer molecule, the second single strand molecule is a target molecule, the first single strand molecule is a probe or primer molecule, the second single strand molecule is a target molecule and the ratio by weight, molarity, number, concentration, or
  • the single strand binding ligand comprises any of a compound which binds to a single strand nucleic acid in a sequence- specific way, a compound which binds to a single strand nucleic acid in a sequence-non ⁇ specific way, a protein, an enzyme, an enzyme which alters the structure of a single strand nucleic acid to which is binds, an enzyme which alters the structure of a single strand nucleic acid to which it binds by breaking or forming a covalent or non-covalent bond, between an atom of the nucleic acid and another atom, an enzyme which cleaves the nucleic acid to which it binds, a restriction enzyme, a restriction endonuclease, an enzyme which alkylates the duplex nucleic acid to which it binds, a nucleic acid ligase, e.g., DNA ligase, an enzyme which promotes or catalyzes the synthesis of a nucleic acid, an enzyme which alters
  • the invention features reaction mixtures including * a plurality of first single strand molecules (e.g., probes, as discussed above), at least one of the second single strand molecules, a single strand binding ligand, and a duplex strand binding ligand (as discussed above), the chemical potential of the single strand binding ligand and the chemical potential of the duplex strand binding ligand being such that the following cycle of events can occur at least n, wherein n is at least 1 , 2, 5, 10, 25, 50, 100, 500, 10 3 , 10 4 , 10 5 or 10 6 under isothermal conditions,
  • the single strand binding ligand is a purified natural product or a non-naturally occurring product; the concentration, number of molecules present, or chemical potential of the single strand binding ligand is greater than the concentration, number of molecules present, or chemical potential of the first single strand, the second single strand, both the first single strand and the second single strand, the duplex, or the combination of the first single strand, the second single strand, and the duplex, e.g., at least n-fold greater, wherein n is at least 1 , 2, 5, 10, 25, 50, 100, 500, 10 3 , 10 4 , lO 5 , or 10 6
  • the single strand binding ligand includes any of a compound which binds to a single strand nucleic acid in a sequence-specific way, a compound which binds to a single strand nucleic acid in a sequence-non-specific way, a protein, an enzyme, an enzyme which alters the structure of a nucleic
  • the concentration, number of molecules present, or chemical potential of the single strand binding ligand is no more than 10 times the concentration, number of molecules present, or chemical potential of the duplex-binding ligand, the concentration, number of molecules present, or chemical potential of the single strand binding ligand is no less than one-tenth the concentration, number of molecules present, or chemical potential of the duplex-binding ligand, the concentration, number of molecules present, or chemical potential of the duplex-binding ligand is no more than 10 times the concentration, number of molecules present, or chemical potential of the single strand binding ligand, the concentration, number of molecules present, or chemical potential of the duplex-binding ligand is no less than one- tenth the concentration, number of molecules present, or chemical potential of the single strand binding ligand
  • the concentration, number of molecules present, or chemical potential of the duplex-binding ligand and the concentration, number of molecules present, or chemical potential ofthe single strand binding ligand are such that the rate of formation of duplex is substantially equal to the rate of formation of single strands
  • the concentration, number of molecules present, or chemical potential of the duplex-binding ligand and the concentration, number of molecules present, or chemical potential of the single strand binding ligand are such that the rate of formation of duplex is no more than 2, 5, 10, 20, 50, 100, 10 3 or 10 4 times the rate of formation of single strands
  • the concentration, number of molecules present, or chemical potential of the duplex-binding ligand and the concentration, number of molecules present, or chemical potential of the single strand binding ligand are such that the rate of formation of single strands is no more than 2, 5 10, 20, 50, 100, 10 3 or 10 4 times the rate of formation of duplex
  • the temperature is below the Tm of the duplex, the temperature is at least n C°, wherein n is 5, 10, 20, 30, 40, 50, 60, 70, or 80, below the Tm, at least one ofthe strand molecules is a purified nucleic acid molecule
  • the number of molecules of, or the chemical potential of, the duplex-binding ligand is greater than the concentration, number of molecules present, or chemical potential of the first single strand, the second single strand, both the first single strand and the second single strand, the duplex, or the combination of the first single strand, the second single strand, and the duplex, e.g., at least n-fold greater, wherein n is at least 1 , 2, 5, 10, 25, 50, 100, 500, 10 3 , 10 4 , 10 5 , or 10 6
  • the first single strand molecule and the single strand molecule are present in substantially equal amounts or at substantially equal chemical potentials in the reaction mix, the first single strand molecule is a probe or primer molecule, the second single strand molecule is a target molecule, the first single strand molecule is a probe or primer molecule, the second single strand molecule is a target molecule and the ratio by weight, molarity, number, concentration, or chemical potentials of the first sequence to the second sequence is less than or equal to 1 1, 2 1, 5 1, 10 1, 25 1 , 50 1 , 100 1 , or 10 n 1 , wherein n is an integer between 3 and 10, inclusive, the ratio by weight, molarity, number, concentration, or chemical potentials of the single strand binding ligand to the single strand in the highest concentration is greater than 1 1 , 2 1 , 5 1, 10 1, 25 1 , 50 1 , 100 1 , or 10 n 1 , wherein n is an integer between 3 and 10, inclusive
  • one or both single strand molecules are DNA, one or both single strand molecules are RNA, one single strand molecule is RNA and the other is DNA, one single strand molecule is a single stranded probe and the other single strand molecule is a target sequence which is to be cleared, detected, or amplified, one single strand molecule is a synthetic, purified natural, genetically engineered, or recombinant DNA or RNA molecule, and the other single strand molecule is a naturally occurring nucleic acid, e.g., a genomic molecule, or chromosome, e.g., a viral, bacterial, plant, or animal nucleic acid, one single strand molecule is a probe and one single strand molecule is the target sequence to be detected or amplified in an amplification reaction, or in a reaction in which amplification or detection is based on the amplification or detection ofthe probe or a fragment ofthe probe
  • the invention features reaction mixtures including * a first single strand molecule (e.g., a probe, as discussed above), a second single strand molecule, a single strand binding ligand, and a duplex-binding ligand (as discussed above), an amount, concentration, or chemical potential of the duplex-binding ligand being sufficient to cause the formation of duplex, and an amount, concentration, or chemical potential of the single strand binding ligand being such that duplex dissociation reaction occurs concurrently with the formation reaction, an amount, concentration, or chemical potentials being such that the rate of duplex formation and the rate of duplex dissociation are such that a cycle of (duplex dissociation)-(single strand binding ligand.
  • a first single strand molecule e.g., a probe, as discussed above
  • a second single strand molecule e.g., a probe, as discussed above
  • a duplex-binding ligand e.g., a duplex binding ligand
  • the cycle can occur least n times under isothermal conditions, wherein n is at least 1, 2, 5, 10, 25, 50, 100, 500, 10 3 , 10 4 , 10 5 , or 106, the single strand binding ligand is a purified natural product or a non-naturally occurring product the concentration, number of molecules present, or chemical potential ofthe single strand binding ligand is greater than the concentration, number of molecules present, or chemical potential of the first single strand, the second single strand, both the first single strand and the second single strand, the duplex, or the combination of the first single strand, the second single strand, and the duplex, e.g., at least n-fold greater, wherein n is at least 1, 2, 5, 10, 25, 50, 100, 500, 10 3 , 10 4 , 10 5 , or 10 6
  • the single strand binding ligand comprises any of a compound which binds to a single strand nucleic acid in a sequence- specific way, a compound which binds to a single strand nucleic acid in a sequence-non ⁇ specific way, a protein, an enzyme, an enzyme which alters the structure of a nucleic acid to which it binds, an enzyme which alters the structure of a nucleic acid to which it binds by breaking or forming a covalent or non-covalent bond, between an atom of the nucleic acid and another atom, an enzyme which cleaves a nucleic acid to which it binds, a restriction enzyme, a restriction endonuclease, an enzyme which methylates the nucleic acid to which it binds, an enzyme which alkylates the duplex nucleic acid to which it binds; an enzyme which alters the primary or secondary structure of a duplex nucleic acid to which it binds, an enzyme which promotes or inhibit
  • the concentration, number of molecules present, or chemical potential of the single strand binding ligand is no more than 10 times the concentration, number of molecules present, or chemical potential of the duplex-binding ligand, the concentration, number of molecules present, or chemical potential of the single strand binding ligand is no less than one-tenth the concentration, number of molecules present, or chemical potential of the duplex-binding ligand, the concentration, number of molecules present, or chemical potential of the duplex-binding ligand is no more than 10 times the concentration, number of molecules present, or chemical potential of the single strand binding ligand, the concentration, number of molecules present, or chemical potential of the duplex-binding ligand is no less than one- tenth the concentration, number of molecules present, or chemical potential of the single strand binding ligand
  • the temperature is below the Tm of the duplex; the temperature is at least n C°, wherein n is 5, 10, 20, 30, 40, 50, 60, 70, or 80, below the Tm; at least one ofthe strand molecules is a purified nucleic acid molecule.
  • the number of molecules of, or the chemical potential of, the duplex-binding ligand is greater than the concentration, number of molecules present, or chemical potential of the first single strand, the second single strand, both the first single strand and the second single strand, the duplex, or the combination of the first single strand, the second single strand, and the duplex, e.g., at least n-fold greater, wherein n is at least 1, 2, 5, 10, 25, 50, 100, 500, 10 3 , 10 4 , 10 5 , or 10 6 .
  • the first single strand molecule and the single strand molecule are present in substantially equal amounts or at substantially equal chemical potentials in the reaction mix; the first single strand molecule is a probe or primer molecule, the second single strand molecule is a target molecule, the first single strand molecule is a probe or primer molecule, the second single strand molecule is a target molecule and the ratio by weight, molarity, number, concentration, or chemical potentials of the first sequence to the second sequence is less than or equal to 1 : 1, 2 1, 5.1, 10: 1, 25: 1, 50.1 , 100.1 , or 10 n 1 , wherein n is an integer between 3 and 10, inclusive.
  • the ratio by weight, molarity, number, concentration, or chemical potentials of the single strand binding ligand to the single strand in the highest concentration is greater than 1 1, 2 1 , 5 1 , 10 1 , 25 1, 50 1 , 100 1, or 10 n . l, wherein n is an integer between 3 and 10, inclusive
  • the single strand binding ligand comprises any of a compound which binds to a single strand nucleic acid in a sequence- specific way, a compound which binds to a single strand nucleic acid in a sequence-non ⁇ specific way, a protein, an enzyme, an enzyme which alters the structure ofa nucleic acid to which it binds, an enzyme which alters the structure of a nucleic acid to which it binds by breaking or forming a covalent or non-covalent bond, between an atom ofthe nucleic acid and another atom, an enzyme which cleaves a nucleic acid to which it binds, a restriction enzyme, a restriction endonuclease, an enzyme which alkylates the duplex nucleic acid to which it binds, a nucleic acid ligase, e.g., DNA ligase, an enzyme which promotes or catalyzes the synthesis of a nucleic acid, an enzyme which alters the primary
  • one or both single strand molecules are DNA, one or both single strand molecules are RNA, one single strand molecule is RNA and the other is DNA, one single strand molecule is a single stranded probe and the other single strand molecule is a target sequence which is to be cleared, detected, or amplified, one single strand molecule is a synthetic, purified natural, genetically engineered, or recombinant DNA or RNA molecule, and the other single strand molecule is a naturally occurring nucleic acid, e.g., a genomic molecule, or chromosome, e.g., a viral, bacterial, plant, or animal nucleic acid, one single strand molecule is a probe and one single strand molecule is the target sequence to be detected or amplified in an amplification reaction, or in a reaction in which amplification or detection is based on the amplification or detection ofthe probe or a fragment ofthe probe
  • Methods of the invention allow detection of one target molecule in a biological sample, detection of a target strand at a concentration of 10" - pMole or less, detection of a target strand at a concentration of 10 " 6 pMole or less, detection of a target strand at a concentration of 10" 7 pMole or less, detection of a target strand at a concentration of 10 " * pMole or less, detection of a target strand at a concentration of 10"9 pMole or less, detection of a target strand at a concentration of 10" ⁇ ⁇ pMole or less; detection of a target strand at a concentration of 10" * ' pMole or less, detection of a target strand at a concentration of 10" 12 pMole or less
  • Reaction fixtures ofthe invention include those in having a target strand concentration of 10 " 5 pMole or less, a target strand concentration of 10"6 pMole or less;
  • Figure 1 illustrates a generalized reaction in which the chemical potentials of a single strand binding agent and a duplex-binding agent provide for cycling between single strand and duplex states Complementary single stranded nucleic acids, S j and S2, react, reversibly, to form a duplex nucleic acid, D
  • This reaction is referred to herein as the basic reaction
  • the free energy for this reaction is designated ⁇ Grj
  • DS-B When agents that preferentially bind duplex (DS-B) are present, an additional free energy (for the reaction wherein DS-B reacts with D to form D:DS-B) contributes to the right hand or (duplex) side of the basic reaction
  • the binding to a duplex of a preferential duplex-binding entity, DS-B has a free energy ⁇ Grjs. ⁇
  • the DS-B may have some affinity for single strand nucleic acid but its affinity for the duplex species must be greater
  • thermodynamic cycling The balance between ⁇ G ⁇ S_B and ⁇ GDS_B determines the net direction of the basic reaction If these two free energies are comparable in value, then an equilibrium situation prevails under which thermodynamic cycling occurs
  • the chemical potentials of SS-B and DS-B are chosen such that ⁇ GSS_B and ⁇ GDS-B are substantially similar in value
  • the arrow on the right in Fig 1 indicates that the chemical potential of the SSB drives duplex form to the single strand form, completing a loop in the cycle
  • the chemical potentials of SS-B and DS-B in a reaction mix will vary with each application but can be chosen empirically
  • the ratios of concentrations of single strand to double strand binding ligand can be determined empirically
  • the concentrations which give thermodynamic cycling can be determined by forming a reaction mixture which includes the single strand species, the duplex, any other necessary reagents (e.g., NTP's in the case of a polymerase catalyzed amplification reaction), and either, the single strand binding ligand, or the duplex-binding ligand, at a predetermined concentration
  • the other binding ligand is then added at progressively increasing (or decreasing) concentrations until thermodynamic cycling is achieved.
  • thermodynamic cycling does not occur the starting concentration of the first binding ligand can be changed and the other binding ligand then added at progressively increasing (or decreasing) concentrations until thermodynamic cycling is achieved
  • Thermodynamic cycling can be recognized by an increase in the reduction of the desired reaction products The increase is often dramatic and can be 10 to 100 fold or more over what is produced in a non-cycling state
  • chemical potentials wherein the single strand binding ligand is lOx above or below that of the double strand binding ligands (or vice versa) will result in thermodynamic cycling
  • concentration of SS-B can be varied to provide for optimal hybridization specificity (as disclosed below).
  • FIG. 2 illustrates a polymerase chain reaction driven by net chemical potential change, also referred to as isothermal PCR
  • single strand primers bind to denatured duplex target to form primer-template complexes (duplexes), with free energy ⁇ G;p
  • DNA polymerase which is a duplex-binding agent
  • polymerase-duplex complexes form in a reaction with free energy ⁇ Grj_r£
  • SS-B single strand binding agents
  • primers and denatured target duplex are bound by those agents to form SS-B-primer complexes and SS-B-target complexes in reactions with free energies ⁇ G gS-B an
  • the absolute chemical potential of the two different nucleic acid binding ligands bound with their substrates is very important in the reactions
  • the actual value for the duplex formation free energy ⁇ Grj is unimportant in the scheme if the chemical potential of each of the ligands for their substrates is high enough to promote duplex -single strand interchange
  • the arrow on the right in Figure 2 indicates that the chemical potential of the SS-B drives duplex form to the single strand form, completing a loop in the cycle
  • thermodynamic cycling will occur, i.e., single strand primers and target molecules will cycle between the single strand and duplex states As strands cycle between single strand and duplex repeated cycles of [primer to target binding (and thus duplex formation), polymerase binding duplex, polymerase catalyzed synthesis of new nucleic acid, and duplex melting] will occur, propelled not by thermal cycling but by thermodynamic cycling
  • polymerase is a DS-B
  • other species of DS-B can be added to the reaction mix to increase the chemical potential of DS-B in the reaction mix DS-B's which do not exhibit substantial inhibition of polymerase can be used
  • the reaction will proceed so vigorously as to produce nonspecific probe target complexes Alternatively, if the polymerase or duplex. polymerase complex concentration or chemical potential is too low, no reaction occurs That is, the enzyme concentration determines duplex-binding and net extent of the reaction, and thereby behaves as a chemical reagent
  • Duplex formation can be modulated by inclusion of a single strand binding agent (SS-B) This affects the left side of the reaction by undergoing a binding reaction with the probe (to form SSB probe) or the target (to form SSB-target) thereby contributing a free energies of single-strand ligand complex ⁇ Gp SS-B anc * ⁇ G-p SS-B
  • the free energy difference between ⁇ Gj £ n ⁇ Gj $S-B and ⁇ Gp sg. ⁇ controls the direction of the cycling equilibrium by mediating duplex formation Note that if under the conditions in which the experiment is performed the free energies ⁇ Grj g and ⁇ Ggs. ⁇ , ⁇ Gj SS-B for the opposing binding reactions are greater than the free energy of duplex formation, ⁇ Grj
  • the chemical potential of SS-B can be chosen to control the fidelity of duplex formation reactions Fidelity of the reactions is independent of initial total DNA concentration when the duplex-binding and single stand binding agents are present at high enough chemical potential A balance between a desired level of fidelity (selectivity) and sensitivity can be provided by choosing the appropriate concentrations of chemical potentials of duplex-binding agents and SS-B. These values are determined empirically or by other methods known in the art Methods employed for describing such compositions are well known in the art
  • the ratio of Taq polymerase(tl ⁇ e duplex-binding ligand) and single stranded binding protein (the single strand binding ligand), were chosen empirically so as to allow thermodynamic cycling in the amplification reaction described below
  • the single strand binding protein in the reaction also serves as the reagent which facilitates thermodynamic cycling of the system General reaction conditions were as follows.
  • Each tube contained 20ng target DNA (300 bp) and primers (lOpM) [one for each strand at lOpM for lOOuL] in a buffer composed of l OOmM TRIS-HC I , pH 7 8, MgCl 2 at 2 5mM, 0.2mM dNTPs; and the enzymes Taq polymerase and SSB
  • the ratio of Taq polymerase to SSB varied as follows Lane 1 0 5 units Taq polymerase, Oug/lOOuL SSB, Lane 2 * 0.5 units Taq polymerase, 0.25 ⁇ g/100uL SSB, Lane 3 0 5 units Taq polymerase, 1.25ug/100uL SSB, Lane 4 0.5 units Taq polymerase, 6.25ug/100uL SSB, Lane 5: 2.5 units Taq polymerase, Oug/lOOuL SSB, Lane 6 2.5 units Taq polymerase, 0.25ug/100uL SSB, Lane 7
  • the ligase chain reaction can be improved by application of methods of the invention.
  • the basic ligase chain reaction involves two probes with sequences that are complementary to adjacent positions in the target
  • the ligase reaction probes and denatured target form a probe-probe:target duplex with a free energy ⁇ G ⁇
  • Agents which preferentially bind to single strands and agents which preferentially bind to duplex can be added to cause thermodynamic cycling and to optimize selectivity
  • the ligase can serve as the duplex-binding ligand Ligase forms a complex with available probe:target duplex, with free energy of ⁇ Grj £
  • the single strand binding ligand forms complexes with the probe and target with free energies ⁇ Gp SS-B an d ⁇ Gj SS-B
  • the actual concentrations of these two enzymes are arrived at empirically and are such that both the forward and the backward reactions proceed with substantially similar rates under prevailing total DNA concentrations As in previous examples, the balance between ⁇

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Abstract

Procédé de réalisation d'une réaction cyclique entre RNasaH et une molécule d'acide nucléique duplex obtenue à partir d'une première molécule (p.ex. une sonde) et d'une seconde molécule (p.ex. une cible) monocaténaire d'acide nucléique, ce procédé consistant à (1) former un mélange réactionnel comprenant une pluralité de premières molécules monocaténaires, au moins une des secondes molécules monocaténaires, un ligand de liaison monocaténaire, et RNaseH, le potentiel chimique du ligand de liaison monocaténaire et le potentiel chimique de RNaseH étant tels que le cycle des évènements pouvant se produire dans des conditions isothermiques est le suivant: (a) formation d'un premier duplex entre un premier brin initial et le second brin, (b) réaction entre RNaseH et le premier duplex formé, (c) dissolution du premier duplex formé, (d) formation d'un second duplex entre un autre premier monobrin et le second brin, et (e) réaction entre RNaseH et le second duplex formé; et (2) permettre que se produisent au moins n cycles parmi les cycles précités, lorsque n est au moins égal à 1, 2, 5, 10, 20, 25, 50, 200, 10?3, 104, 105 ou 106¿.
PCT/US1996/015199 1995-09-22 1996-09-23 Reactions d'acide nucleique WO1997011199A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999001572A2 (fr) * 1997-07-03 1999-01-14 Id Biomedical Corporation Procedes permettant de detecter rapidement les staphyloccoques resistant a la methicilline
WO1999001569A2 (fr) * 1997-07-03 1999-01-14 Id Biomedical Corporation Additifs pour reactions de sondes en cycles

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995000667A1 (fr) * 1993-06-17 1995-01-05 The Research Foundation Of State University Of New York Procedes ameliores pour detecter des sequences d'acides nucleiques
WO1995000666A1 (fr) * 1993-06-17 1995-01-05 The Research Foundation Of State University Of New York Ameliorations des reactions des acides nucleiques

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995000667A1 (fr) * 1993-06-17 1995-01-05 The Research Foundation Of State University Of New York Procedes ameliores pour detecter des sequences d'acides nucleiques
WO1995000666A1 (fr) * 1993-06-17 1995-01-05 The Research Foundation Of State University Of New York Ameliorations des reactions des acides nucleiques

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999001572A2 (fr) * 1997-07-03 1999-01-14 Id Biomedical Corporation Procedes permettant de detecter rapidement les staphyloccoques resistant a la methicilline
WO1999001569A2 (fr) * 1997-07-03 1999-01-14 Id Biomedical Corporation Additifs pour reactions de sondes en cycles
WO1999001572A3 (fr) * 1997-07-03 1999-03-25 Id Biomedical Corp Procedes permettant de detecter rapidement les staphyloccoques resistant a la methicilline
WO1999001569A3 (fr) * 1997-07-03 1999-03-25 Id Biomedical Corp Additifs pour reactions de sondes en cycles
US6136533A (en) * 1997-07-03 2000-10-24 Id Biomedical Additives for use in cycling probe reactions
US6503709B1 (en) 1997-07-03 2003-01-07 Id Biomedical Corporation Methods for rapidly detecting methicillin resistant staphylococci

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